PS tiene una Cátedra de investigación de Canadá en Biología Celular de la retina y el Premio del Instituto de Investigación Alcon Nueva investigador. Este trabajo fue apoyado por becas de los Institutos Canadienses de Investigación en Salud (221,478), la Asociación Canadiense de Diabetes (OG-3-11-3329-PS), las Ciencias Naturales e Ingeniería de Investigación de Canadá (418.637) y la Fundación Lucha contra la Ceguera Canadá. El apoyo también fue proporcionado por el Réseau de Recherche en Santé de la Vision du Québec.

Declaración de Ética: Todos los experimentos con animales se adhiere a los lineamientos de cuidado animal establecidas por la Asociación para la Investigación en Visión y Oftalmología (ARVO) Declaración para el uso de animales en Oftálmica y Visión Investigación y el Consejo Canadiense de los Animales. 1. Oxígeno inducido Retinopatía (OIR) <ol><li> Fecha de apunte de nacimiento de crías de ratón como P0. </li><li> Registre todos los pesos de los animales a la entrada en O 2 para asegurar una variedad de peso adecuado. Nota: Para C57BL / 6 ratones a P17, peso corporal debe oscilar entre 5 y 7,5 g para NV máxima 32. Con el fin de mantener la coherencia del medio ambiente, se recomienda utilizar como compañeros de camada de control (para los ratones modificados genéticamente, así como los ratones que recibieron tratamientos experimentales). Al evaluar los efectos de un vector viral, se debe considerar el tropismo del virus y dar tiempo suficiente para la plena expresión de transgenes viralmente entregados. Viral rápidamente expresandovectores tales como los lentivirus 3 ª generación 24, 25, se recomiendan 33. </li><li> Cachorros Place ratón en P7 (C57BL / 6 o deseado cepa) y una CD1 fomentar madre en una cámara de oxígeno fijado en el 75% de O 2 durante 5 días 27. La humedad del ambiente y la temperatura se mantienen constantes a lo largo de O 2 de la exposición. Nota: Las instalaciones de investigación equipados con una fuente central de O 2 son ideales y limitan la sustitución engorroso de O 2 tanques vacíos. Si se trabaja con ratones manipulados genéticamente, es importante asegurarse de que los ratones control y experimental se adquieren del mismo proveedor para limitar las derivas genéticas dentro de las mismas cepas 30, 29. </li><li> En P12, eliminar a los ratones de la cámara de oxígeno y devolver los animales al ambiente de O 2. </li></ol> 2. Inyección intravítrea de Entrega de los compuestos de la retina interna (Whes evaluar los efectos de un compuesto farmacológico o Proteína Recombinante) <ol><li> En P14, anestesiar a los ratones con 2% de isoflurano en oxígeno 2 L / min (o comité de protección de los animales aprobado anestésico de elección). Con el fin de verificar la eficacia de la anestesia, secuencialmente pellizcar la cola, pata trasera y el oído con pinzas. </li><li> Coloca el ratón sobre su vientre. </li><li> Usando una jeringa estéril de 10 l equipado con una aguja de vidrio tirado biselado, realizar una inyección de un volumen máximo de 1 l de solución que contiene el compuesto que está siendo investigado o vehículo (solución salina fisiológica) en el limbo posterior del ojo, con un 45 ° ángulo de evitar la lente. Nota: El-aguja de vidrio arrastrado está unido a la jeringa utilizando una gota de resina epoxi. </li><li> Aplique una gota de ungüento oftálmico lubricante (idealmente con antibióticos) con un hisopo para el ojo del ratón. </li><li> Devuelva el ratón de nuevo a la jaula con el fomento de la madre. Los ratones son entonces monitoreados cuidadosamente hasta que recubierto y totalmente ambulatoria. </li></ol> 3. Evaluación de la perfusión de buques y la función de barrera (Integridad) mediante angiografía con fluoresceína <ol><li> Anestesie ratones con 2% de isoflurano en oxígeno 2 L / min (o comité de protección de los animales aprobado anestésico de elección). Con el fin de verificar la eficacia de la anestesia, secuencialmente pellizcar la cola, pata trasera y el oído con pinzas. Nota: Esto se realiza típicamente a P17 cuando se evalúa la regeneración. También llevar-a cabo el análisis en P19 y P21 para determinar si la integridad vascular se conserva en el tiempo. </li><li> Una vez anestesiado, se pesa el ratón. </li><li> Haga una incisión en la línea media del abdomen con unas tijeras de disección. Nota: Los instrumentos de disección se deben revisar y afilados con regularidad. </li><li> Cortar las costillas lateralmente y elevar la caja torácica con la ayuda de fórceps. Nota: Es necesario cortar como lateralmente como sea posible para evitar daños en el corazón. </li><li> Después de retirar periptejido heral desde el corazón, pinza la aorta descendente con unas pinzas hemostáticas. </li><li> Inyecte lentamente con fluoresceína dextrano al ventrículo izquierdo con una aguja 25. Nota: Si la función de barrera vascular se investiga, se emplea 70 kDa fluoresceína-dextrano, ya que se saldrá de los vasos cuando se ve comprometida la integridad del vaso. Si el investigador quiere lanzar vasos sanguíneos, se utiliza 2 MDa fluoresceína-dextrano. Los pasos críticos: 1) Para asegurar un reparto homogéneo, centrifugar fluoresceína dextrano e inyectar el sobrenadante, 2) Con el fin de prevenir la constricción de vasos, inyectar solución de fluoresceína dextrano calentado, 3) su tiempo de circulación no debe exceso de 4 min. </li><li> Decapitar a los ratones 2 min después de la inyección con unas tijeras de funcionamiento. </li></ol> 4. Enucleación y fijación de los ojos Nota: Al evaluar las posibilidades de regeneración vascular, primero recoger retinas en P12 y P14 y, además, al P17. Aumentar el número de punto en el tiempo de muestreos para una determinación más precisa de las tasas de revascularización 24. <ol><li> Incline la cabeza del ratón y colocarlo en su lado. </li><li> Quite la piel y los párpados cubren los ojos con unas tijeras de disección. </li><li> Coloque fórceps curvados debajo del ojo y suavemente tire hacia arriba hasta que el nervio óptico se corta. </li><li> Gire la cabeza del ratón hacia el otro lado y realizar los mismos pasos (pasos 4.2 y 4.3). </li><li> Para asegurar una mejor penetración de fijador, perforar un agujero en la cámara anterior del ojo utilizando aguja de 30 g. </li><li> Transferencia de ojos a un tubo que contiene 4% de paraformaldehído (PFA) y fijar durante 1 hora a temperatura ambiente. </li><li> Eliminar PFA y lavar los ojos 4 veces con una solución de PBS enfriado en hielo. </li></ol> 5. Disección de retina <ol><li> Coloque los ojos de ratón en una placa de Petri que contiene PBS frío y realizar la disección de las retinas bajo un microscopio estereoscópico. </li><li> Quite la grasa / tejido adicional que rodea el ojo con ciencia microdisecciónssors. </li><li> Cortar la córnea con tijeras micro-disección. </li><li> El uso de dos pares de pinzas, minuciosamente la cáscara de la esclerótica de distancia desde la periferia hacia el nervio óptico y desechar. </li><li> Apriete la lente (bola blanquecina debajo de la córnea) con unas pinzas y extraerlo de la caja del visor. Use un par de pinzas como soporte, y la otra para agarrar y levantar con cuidado y retire el objetivo. </li><li> Separar los vasos hialoideos desde el lado interior de la retina utilizando cepillos pequeños (tamaño 0) y fórceps. </li><li> Retire los bloques de vasos hyaloid conectados al disco óptico utilizando fórceps. </li><li> Transferencia diseccionó la retina a 2 ml de tubos de microcentrífuga que contiene PBS y lugar en hielo antes de comenzar el procedimiento de tinción. </li></ol> 6. Retiniana vascular tinción <ol><li> Incubar las retinas disecadas durante la noche con agitación suave a 4 ° C en una solución de fluorescencia B4-isolectina acoplado (rodamina-lectina u otra) en PBS que contenía 1 mM de CaCl2 </sub&gt; (se recomienda una dilución de una solución B4 2 mg / ml isolectina 1:100). Durante todo el procedimiento de tinción, cubrir los tubos con papel de aluminio o una lámina opaca para protegerlo de la luz. </li><li> Al día siguiente, eliminar la solución de tinción y lavar las retinas 3x en PBS durante 10 min a temperatura ambiente. </li></ol> 7. Preparación de la retina Flatmounts <ol><li> Traslado retinas, del lado de los fotorreceptores hacia abajo, en un portaobjetos de microscopio y hacer cuatro profundas incisiones radiales equidistantes utilizando un bisturí quirúrgico para dividir la retina en cuatro cuadrantes de igual tamaño. Durante las incisiones, prepararse la retina con un cepillo de modo que no se mueve. </li><li> El uso de dos cepillos empapados en PBS, aplane cuidadosamente la cuadrantes fotorreceptor boca abajo y sumergir la retina en medio de montaje para evitar la foto-blanqueo. A continuación, coloque cuidadosamente un cubreobjetos sobre la superficie de la retina montado sin la aplicación de presión y asegurarse de que las burbujas de aire no se acumulan debajo de la hoja de la cubierta. &lt;/ Li&gt; </ol> 8. Imaging y Cuantificación de Vasoobliteration (VO) y neovascularización (NV), como se ha descrito previamente 31 <ol><li> Tomar imágenes de retinas-todo ello montado con un microscopio de epi-fluorescencia con un aumento de 10X. </li><li> Abre la imagen en la retina en software de edición fotográfica, hilvanar y medir el área total de la retina, y la zona avascular. Área puede expresarse en píxeles. </li><li> Determinar la extensión del VO dividiendo el número de píxeles en la zona avascular por el número de píxeles en el área total de la retina. </li><li> Determinar la extensión de NV dividiendo el número de píxeles de NV por el número de píxeles en el área total de la retina tal como se describe 31. </li></ol>

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G., Jiang, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogenesis,+angiogenesis,+and+MRI+indices+of+functional+recovery+from+stroke.">Neurogenesis, angiogenesis, and MRI indices of functional recovery from stroke.</a> Stroke. 38, 827-831 (2007).</li><li>Li, L., 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=Angiogenesis+and+improved+cerebral+blood+flow+in+the+ischemic+boundary+area+detected+by+MRI+after+administration+of+sildenafil+to+rats+with+embolic+stroke.">Angiogenesis and improved cerebral blood flow in the ischemic boundary area detected by MRI after administration of sildenafil to rats with embolic stroke.</a> Brain Res. 1132, 185-192 (2007).</li><li>Robinson, R., Barathi, V. A., Chaurasia, S. S., Wong, T. Y., Kern, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Update+on+animal+models+of+diabetic+retinopathy:+from+molecular+approaches+to+mice+and+higher+mammals.">Update on animal models of diabetic retinopathy: from molecular approaches to mice and higher mammals.</a> Dis Model Mech. 5, 444-456 (2012).</li><li>Chia, R., Achilli, F., Festing, M. F., Fisher, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origins+and+uses+of+mouse+outbred+stocks.">The origins and uses of mouse outbred stocks.</a> Nat Genet. 37, 1181-1186 (2005).</li><li>Jenuth, J. P., Peterson, A. C., Shoubridge, 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=Tissue-specific+selection+for+different+mtDNA+genotypes+in+heteroplasmic+mice.">Tissue-specific selection for different mtDNA genotypes in heteroplasmic mice.</a> Nat Genet. 16, 93-95 (1997).</li><li>Mattapallil, M. J., 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+Rd8+mutation+of+the+Crb1+gene+is+present+in+vendor+lines+of+C57BL/6N+mice+and+embryonic+stem+cells,+and+confounds+ocular+induced+mutant+phenotypes.">The Rd8 mutation of the Crb1 gene is present in vendor lines of C57BL/6N mice and embryonic stem cells, and confounds ocular induced mutant phenotypes.</a> Investigative ophthalmolog., &amp; visual science. 53, 2921-2927 (2012).</li></ol>

Los autores no tienen nada que revelar.

¿Cuál es la forma más eficaz para estimular el crecimiento de nuevos vasos sanos en el tejido nervioso isquémico? ¿Es terapéuticamente válida para interferir con y acelerar origen natural recrecimiento vascular? En patologías neuro-isquémica como retinopatías isquémicas o derrame cerebral, degeneración vascular se asocia con una reducción de la función neuronal 35-38. Por lo tanto para hacer frente a la lesión inicial, el restablecimiento de la microcirculación regional durante la fase inmedia...

A lo largo del desarrollo del SNC, los nervios, las células inmunes y los vasos sanguíneos establecer redes notablemente acoplados a garantizar una adecuada perfusión tisular y permitir la transmisión de información sensorial 1-5. El detalle de los resultados de los sistemas vasculares en la oxigenación tisular insuficiente y suministro metabólico comprometido y está cada vez más reconocido como un importante contribuyente a la patogénesis de las enfermedades neurodegenerativas 6. Deserción vascular y el deterioro de la unidad neurovascular dentro del cerebro, por ejemplo, se asocia con demencia vascular, lesiones vasculares de la materia blanca del cerebro 7 y la enfermedad de Alzheimer con estenosis de las arteriolas y los vasos pequeños 8. Además, se cree alteración de la función barrera vascular para contribuir esclerosis múltiple 9 y la esclerosis lateral amiotrófica 10. De importancia directa para el modelo de la retina se describe en este protocolo, el cegamientoenfermedades tales como la retinopatía diabética 11 y la retinopatía de la prematuridad 12, 13 se caracterizan por una fase de degeneración vascular temprano. El estrés isquémico posterior sobre la retina neurovascular desencadena una segunda fase de la neovascularización excesiva y patológica que probablemente se origina como una respuesta compensatoria a oxígeno restablecerá y suministro de energía 14-16. Una estrategia atractiva para superar el estrés isquémico que es central a la progresión de la enfermedad es restaurar redes vasculares funcionales específicamente en las zonas isquémicas de la neuro-retina (Figuras 2 y 3). Provocar una respuesta angiogénica controlada puede venir a través como contra-intuitivo para una condición en la que los tratamientos anti-angiogénicos tales como anti-VEGF son considerados como tratamientos adaptados. Sin embargo, la evidencia de la validez de este enfoque es el montaje. Por ejemplo, la mejora de recrecimiento vascular &quot;fisiológica-like&quot; en ischemretinopatías ic se ha demostrado elegantemente a través de la introducción de las células precursoras endoteliales 17, la inhibición de VEGF de células de Müller-expresado regulación a la baja inducida de otros factores angiogénicos, 18 de inyección de progenitores mieloides 19, la inhibición de NADPH oxidasa inducida por la apoptosis 20, el aumento de la dieta ω-3 grasos poliinsaturados la ingesta de ácido 21, el tratamiento con un fragmento carboxilo-terminal de triptófano ARNt sintetasa 22, y la administración directa de VEGF o FGF-2 para la protección de las células gliales 23. Por otra parte, hemos demostrado que la modulación de las señales de orientación neuronales clásicas tales como Semaphorins o netrinas en retinopatías isquémicas acelera la regeneración vascular de los vasos sanos dentro de la retina y, en consecuencia reduce la angiogénesis patológica 24, 25. De relevancia clínica directa, varios de los estudios en animales antes mencionados proporcionan evidencia de que la promoción de re vasculargeneración durante la fase isquémica temprana de retinopatías puede reducir significativamente la neovascularización que ponen en peligro la vista previa de la retina 19, 23, 24, 26, probablemente a través de la reducción de la carga isquémica. La elaboración de estrategias terapéuticas que estimulan la regeneración de los vasos funcionales sigue siendo un reto importante para los biólogos vasculares. Aquí se describe un sistema experimental que emplea el modelo de ratón de la retinopatía inducida por oxígeno (OIR) para explorar estrategias para modular el recrecimiento vascular en la retina. Desarrollado por Smith et al. En 1994 27, este modelo sirve como sustituto de las retinopatías proliferativas humanas y consiste en la exposición de las crías P7 ratón al 75% de O 2 hasta el P12 y, posteriormente, la reintroducción de las crías al ambiente de la sala O2-tensión (Figura 1). Este paradigma imita débilmente un escenario en el que se ventila un bebé prematurocon O 2. La exposición de las crías de ratón a hiperoxia provoca degeneración de los capilares de la retina y de la microvasculatura, y produce una superficie reproducible de vaso-obliteración (VO) suele evaluarse a la salida de O 2 en P12, aunque el área de VO máxima se alcanza a las 48 horas (P9) después exposición a O 2 28. En el ratón, las zonas avasculares VO regeneran espontáneamente en el transcurso de la semana siguiente a la reintroducción de aire de la habitación y finalmente zonas VO están completamente re-vascularizados (Figura 2). La reintroducción de aire de la sala de ratones sometidos a OIR también provoca la neovascularización de la retina antes de la (NV) (máximo a P17) que se evalúa típicamente para determinar la eficacia de los paradigmas de tratamiento anti-angiogénicos. En su forma más pura, el modelo OIR proporciona una herramienta altamente reproducible y cuantificable para evaluar la degeneración vascular inducida por el oxígeno y determinar el grado de destrucción neovascularización pre-retinal 29-31. <p class = &quot;jove_content&quot;&gt; Varios paradigmas de tratamiento exploratorio que modulan la regeneración vascular del SNC puede ser investigada mediante el modelo de OIR, incluyendo el uso de los compuestos farmacológicos, la terapia génica, la supresión de genes y más. La propensión de un enfoque dado para influir en el nuevo crecimiento vascular se evaluó paso a paso en la ventana entre P12 (VO máxima después de la salida de la hiperoxia) y P17 (NV máxima). Evaluación de los resultados del tratamiento en NV patológica se puede determinar rápidamente y fácilmente en paralelo y ha sido descrita a fondo por Stahl y colegas 30, 31. Aquí le ofrecemos una forma sencilla y paso a paso para investigar la modulación de revascularización fisiológica dentro de la retina neural por compuestos farmacológicos, terapéuticos potenciales, vectores virales o para estudiar la influencia de los genes candidatos en los ratones manipulados genéticamente.

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			<td height="21" style="height:21px;width:639px;">C57Bl/6 mice ((Other strains may be used; angiogenic response varies from one strain to the other)</td>
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			<td height="38" style="height:38px;width:639px;">CD1 nursing mothers</td>
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			<td height="42" style="height:42px;width:639px;">Dissecting Scissors straight</td>
			<td style="width:181px;">World Precision Instruments</td>
			<td style="width:175px;">14393</td>
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			<td height="42" style="height:42px;width:639px;">Iris Forceps, curved, serrated</td>
			<td style="width:181px;">World Precision Instruments</td>
			<td style="width:175px;">15915</td>
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			<td height="21" style="height:21px;width:639px;">Brushes 362R size 0</td>
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			<td height="42" style="height:42px;width:639px;">Dumont Forceps #3; straight</td>
			<td style="width:181px;">World Precision Instruments</td>
			<td style="width:175px;">500338</td>
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			<td height="21" style="height:21px;width:639px;">Rhodamine Griffonia (Bandeiraea) Simplicifolia Lectin I</td>
			<td style="width:181px;">Vector Laboratories, Inc</td>
			<td style="width:175px;">RL-1102</td>
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			<td height="21" style="height:21px;width:639px;">Microscope slides</td>
			<td style="width:181px;">VWR</td>
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			<td height="42" style="height:42px;width:639px;">Fluoromount G</td>
			<td style="width:181px;">Electron Microscopy Sciences</td>
			<td style="width:175px;">17984-25</td>
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			<td height="21" style="height:21px;width:639px;">Zeiss Axio Observer Z1 Inverted Phase and Fluorescence Microscope</td>
			<td style="width:181px;">Zeiss</td>
			<td style="width:175px;"></td>
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			<td height="42" rowspan="2" style="height:42px;width:639px;">Leica MZ9.5 Stereomicroscope</td>
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			<td height="21" style="height:21px;width:639px;">Fluorescein isothicyanate-dextran, 70000</td>
			<td style="width:181px;">Sigma-Aldrich</td>
			<td style="width:175px;">46945</td>
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assessment of vascular regeneration in the cns using the mouse retina

La retina de roedor es tal vez el sistema de mamífero más accesible en el que para investigar la interacción neurovascular dentro del sistema nervioso central (SNC). Se está reconociendo cada vez más que algunas enfermedades neurodegenerativas como el Alzheimer, la esclerosis múltiple y la esclerosis lateral amiotrófica elementos presentes de compromiso vascular. Además, las causas más importantes de ceguera en la población de edad pediátrica y de trabajo (retinopatía del prematuro y la retinopatía diabética, respectivamente) se caracterizan por una degeneración vascular y la insuficiencia de recrecimiento vascular fisiológico. El objetivo de este documento técnico es proporcionar un protocolo detallado para estudiar la regeneración vascular del SNC en la retina. El método puede ser empleado para dilucidar los mecanismos moleculares que conducen a la insuficiencia de crecimiento vascular después de la lesión isquémica. Además, las posibles modalidades terapéuticas para acelerar y restaurar plexos vasculares sanas se pueden explorar. Hallazgos obtaineD utilizando el enfoque descrito puede proporcionar vías terapéuticas para retinopatías isquémicas tales como el de la diabetes o la prematuridad y posiblemente beneficiar a otros trastornos vasculares del SNC.

El modelo OIR es ampliamente utilizado para estudiar la degeneración vascular inducida por el oxígeno y la neovascularización patológica inducida por isquemia en la retina y ha sido instrumental en el desarrollo de tratamientos anti-angiogénicos que trabajan actualmente para las enfermedades oculares 27, 29, 30. Los resultados obtenidos con este modelo pueden ser libremente extrapolar a retinopatías isquémicas tales como la retinopatía diabética proliferativa y retinopatía del prematuro 30.</su...

Watch this Scientific Journal Video about Assessment of Vascular Regeneration in the CNS Using the Mouse Retina at JoVE.com

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

La retina de roedores ha sido reconocida como una ventana de acceso al cerebro. En este documento técnico se proporciona un protocolo que emplea el modelo de ratón de retinopatía inducida por oxígeno para estudiar los mecanismos que conducen al fracaso de la regeneración vascular en el sistema nervioso central después de la lesión isquémica. El sistema descrito también se puede aprovechar para explorar estrategias para promover el nuevo crecimiento de vasos sanguíneos funcionales dentro de la retina y sistema nervioso central.

Evaluación de la Vascular regeneración en el SNC Utilizando la retina del ratón

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). ...

assessment-of-vascular-regeneration-in-the-cns-using-the-mouse-retina

Research

JoVE Journal

Neuroscience

14.3K vistas.  McGill University.  La retina de roedores ha sido reconocida como una ventana de acceso al cerebro. En este documento técnico se proporciona un protocolo que emplea el modelo de ratón de retinopatía inducida por oxígeno para estudiar los mecanismos que conducen al fracaso de la regeneración vascular en el sistema nervioso central después de la lesión isquémica. El sistema descrito también se puede aprovechar para explorar estrategias para promover el nuevo crecimiento de vasos sang...

Video: Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to cis-regulatory elements (CREs), short (&tilde;300-600 bp) stretches of genomic DNA which can lie upstream, downstream, or within the introns of the genes they control. CREs (i.e., enhancers/promoters) typically consist of multiple clustered binding sites for both transcriptional activators and repressors1-3. They serve as logical integrators of transcriptional input giving a unitary output in the form of spatiotemporally precise and quantitatively exact promoter activity. Most studies of mammalian cis-regulation to date have relied on mouse transgenesis as a means of assaying the enhancer function of CREs4-5. This technique is time-consuming, costly and, on account of insertion site effects, largely non-quantitative. On the other hand, quantitative assays for mammalian CRE function have been developed in tissue culture systems (e.g., dual luciferase assays), but the in vivo relevance of these results is often uncertain. 
Electroporation offers an excellent alternative to traditional mouse transgenesis in that it permits both spatiotemporal and quantitative assessment of cis-regulatory activity in living mammalian tissue. This technique has been particularly useful in the analysis of cis-regulation in the central nervous system, especially in the cerebral cortex and the retina6-8. While mouse retinal electroporation, both in vivo and ex vivo, has been developed and extensively described by Matsuda and Cepko6-7,9, we have recently developed a simple approach to quantify the activity of photoreceptor-specific CREs in electroporated mouse retinas10. Given that the amount of DNA that is introduced into the retina by electroporation can vary from experiment to experiment, it is necessary to include a co-electroporated 'loading control' in all experiments. In this respect, the technique is very similar to the dual luciferase assay used to quantify promoter activity in cultured cells. 
When assaying photoreceptor cis-regulatory activity, electroporation is usually performed in newborn mice (postnatal day 0, P0) which is the time of peak rod production11-12. Once retinal cell types become post-mitotic, electroporation is much less efficient. Given the high rate of rod birth in newborn mice and the fact that rods constitute more than 70% of the cells in the adult mouse retina, the majority of cells that are electroporated at P0 are rods. For this reason, rod photoreceptors are the easiest retinal cell type to study via electroporation. The technique we describe here is primarily useful for quantifying the activity of photoreceptor CREs.

Quantifying the Activity of cis-Regulatory Elements in the Mouse Retina by Explant Electroporation

This protocol describes a simple and inexpensive way to quantify the activity of cis-regulatory elements (i.e., enhancer/promoters) in living mouse retinas via explant electroporation. DNA preparation, retinal dissection, electroporation, retinal explant culture, and post-fixation analysis and quantification are described.

La cuantificación de la actividad de Cis Elementos reguladores en la retina del ratón por electroporación explante

Transcription factors within cellular gene networks control the spatiotemporal pattern and levels of expression of their target genes by binding to ...

Investigación

Neurociencias

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

En electroporación in vivo de la retina del ratón en desarrollo

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

Fast Micro-iontoforesis de glutamato y GABA: una herramienta útil para investigar la integración sináptica

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

The Corneal Micropocket Assay: A Model of Angiogenesis in the Mouse Eye

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets containing specific growth factors that trigger blood vessel growth throughout the naturally avascular cornea, angiogenesis can be measured and quantified. In this assay the angiogenic response is generated over the course of several days, depending on the type and dose of growth factor used. The induction of neovascularization is commonly triggered by either basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF). By combining these growth factors with sucralfate and hydron (poly-HEMA (poly(2-hydroxyethyl methacrylate))) and casting the mixture into pellets, they can be surgically implanted in the mouse eye. These uniform pellets slowly-release the growth factors over five or six days (bFGF or VEGF respectively) enabling sufficient angiogenic response required for vessel area quantification using a slit lamp. This assay can be used for different applications, including the evaluation of angiogenic modulator drugs or treatments as well as comparison between different genetic backgrounds affecting angiogenesis. A skilled investigator after practicing this assay can implant a pellet in less than 5 min per eye.

The protocol describes the corneal micropocket assay as developed in mice.

El microbolsa Ensayo corneal: un modelo de angiogénesis en el ojo del ratón

The mouse corneal micropocket assay is a robust and quantitative in vivo assay for evaluating angiogenesis. By using standardized slow-release pellets ...

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to blindness in many retinal diseases. Calcium (Ca2+), a key second messenger in photoreceptor signaling and metabolism, has been proposed to be indirectly linked with photoreceptor degeneration in various animal models. Systematically studying these aspects of cone physiology and pathophysiology has been hampered by the difficulties of electrically recording from these small cells, in particular in the mouse where the retina is dominated by rod photoreceptors. To circumvent this issue, we established a two-photon Ca2+ imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca2+ biosensor TN-XL exclusively in cones and can be crossbred with mouse models for photoreceptor degeneration. The protocol described here involves preparing vertical sections (&#8220;slices&#8221;) of retinas from mice and optical imaging of light stimulus-evoked changes in cone Ca2+ level. The protocol also allows &#8220;in-slice measurement&#8221; of absolute Ca2+ concentrations; as the recordings can be followed by calibration. This protocol enables studies into functional cone properties and is expected to contribute to the understanding of cone Ca2+ signaling as well as the potential involvement of Ca2+ in photoreceptor death and retinal degeneration.

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

We describe a protocol to monitor Ca2+ dynamics in the axon terminals of cone photoreceptors using an ex-vivo&#160;slice preparation of the mouse retina. This protocol allows comprehensive studies of cone Ca2+ signaling in an important mammalian model system, the mouse.

Imagen Ca<sup&gt; 2 +</sup&gt; Dinámica de Terminales cono fotorreceptor axón de la retina del ratón

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

Using the Electroretinogram to Assess Function in the Rodent Retina and the Protective Effects of Remote Limb Ischemic Preconditioning

The ERG is the sum of all retinal activity. The ERG is usually recorded from the cornea, which acts as an antenna that collects and sums signals from the retina. The ERG is a sensitive measure of changes in retinal function that are pan-retinal, but is less effective for detecting damage confined to a small area of retina. In the present work we describe how to record the &#8216;flash&#8217; ERG, which is the potential generated when the retina is exposed to a brief light flash. We describe methods of anaesthesia, mydriasis and corneal management during recording; how to keep the retina dark adapted; electrode materials and placement; the range and calibration of stimulus energy; recording parameters and the extraction of data. We also describe a method of inducing ischemia in one limb, and how to use the ERG to assess the effects of this remote-from-the-retina ischemia on retinal function after light damage. A two-flash protocol is described which allows isolation of the cone-driven component of the dark-adapted ERG, and thereby the separation of the rod and cone components. Because it can be recorded with techniques that are minimally invasive, the ERG has been widely used in studies of the physiology, pharmacology and toxicology of the retina. We describe one example of this usefulness, in which the ERG is used to assess the function of the light-damaged retina, with and without a neuroprotective intervention; preconditioning by remote ischemia.

The electroretinogram (ERG) is an electrical potential generated by the retina in response to light. This paper describes how to use the ERG to assess retinal function, in dark-adapted rats, and how it can be can be used to assess a neuroprotective intervention, in the present case remote ischemic preconditioning.

Utilizando el Electrorretinograma para evaluar la función en el Roedor Retina y los efectos protectores de distancia Limb Preacondicionamiento Isquémico

The ERG is the sum of all retinal activity. The ERG is usually recorded from the cornea, which acts as an antenna that collects and sums signals from the ...

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the dorsal root ganglion (DRGs) extends a single axon with two branches. One branch goes to the peripheral nerve (peripheral branch), and the other branch enters the spinal cord through the dorsal root (central branch). After the neural injury, the peripheral branch regenerates robustly whereas the central branch does not regenerate. Due to the high regenerative capacity, sensory axon regeneration has been widely used as a model system to study mammalian axon regeneration in both the peripheral nervous system (PNS) and the central nervous system (CNS). Here, we describe a previously established approach protocol to manipulate gene expression in mature sensory neurons in vivo via electroporation. Based on transfection with plasmids or small RNA oligos (siRNAs or microRNAs), the approach allows for both loss- and gain-of-function experiments to study the roles of genes-of-interests or microRNAs in regulation of axon regeneration in vivo. In addition, the manipulation of gene expression in vivo can be controlled both spatially and temporally within a relatively short time course. This model system provides a unique tool to investigate the molecular mechanisms by which mammalian axon regeneration is regulated in vivo.

Investigating Mammalian Axon Regeneration: In Vivo Electroporation of Adult Mouse Dorsal Root Ganglion

Electroporation is an effective approach to deliver genes of interests into cells. By applying this approach in vivo on the neurons of adult mouse dorsal root ganglion (DRG), we describe a model to study axon regeneration in vivo.

Investigadora de axón mamífero de regeneración: En Vivo electroporación de ganglio de raíz Dorsal de ratón adulto

Electroporation is an essential non-viral gene transfection approach to introduce DNA plasmids or small RNA molecules into cells. A sensory neuron in the ...

The Power of Interstimulus Interval for the Assessment of Temporal Processing in Rodents

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in neurocognitive disorders. Despite the popularization of prepulse inhibition (PPI) in recent years, many current protocols promote using a percent of control measure, thereby precluding the assessment of temporal processing. The present study used cross-modal PPI and gap prepulse inhibition (gap-PPI) to demonstrate the benefits of employing a range of interstimulus intervals (ISIs) to delineate effects of sensory modality, psychostimulant exposure, and age. Assessment of sensory modality, psychostimulant exposure, and age reveals the utility of an approach varying the interstimulus interval (ISI) to establish the shape of the ISI function, including increases (sharper curve inflections) or decreases (flattening of the response amplitude curve) in startle amplitude. Additionally, shifts in peak response inhibition, suggestive of a differential sensitivity to the manipulation of ISI, are often revealed. Thus, the systematic manipulation of ISI affords a critical opportunity to evaluate temporal processing, which may reveal the underlying neural mechanisms involved in neurocognitive disorders.

Temporal processing, a preattentive process, may underlie deficits in higher-level cognitive processes, including attention, commonly observed in neurocognitive disorders. Using prepulse inhibition as an exemplar paradigm, we present a protocol for manipulating interstimulus interval (ISI) to establish the shape of the ISI function to provide an assessment of temporal processing.

El poder del Interstimulus intervalo para la evaluación de proceso Temporal en roedores

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in ...

Horizontal Hippocampal Slices of the Mouse Brain

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as well as the manifestation of severe brain disorders, including Alzheimer&#8217;s disease and epilepsy. The hippocampus receives a high degree of intra- and inter-connectivity, securing a proper communication with internal and external brain structures. This connectivity is accomplished via different informational flows in the form of fiber pathways. Brain slices are a frequently used methodology when exploring neurophysiological functions of the hippocampus. Hippocampal brain slices can be used for several different applications, including electrophysiological recordings, light microscopic measurements as well as several molecular biological and histochemical techniques. Therefore, brain slices represent an ideal model system to assess protein functions, to investigate pathophysiological processes involved in neurological disorders as well as for drug discovery purposes.
There exist several different ways of slice preparations. Brain slice preparations with a vibratome allow a better preservation of the tissue structure and guarantee a sufficient oxygen supply during slicing, which present advantages over the traditional use of a tissue chopper. Moreover, different cutting planes can be applied for vibratome brain slice preparations. Here, a detailed protocol for a successful preparation of vibratome-cut horizontal hippocampal slices of mouse brains is provided. In contrast to other slice preparations, horizontal slicing allows to keep the fibers of the hippocampal input path (perforant path) in a fully intact state within a slice, which facilitates the investigation of entorhinal-hippocampal interactions. Here, we provide a thorough protocol for the dissection, extraction, and acute horizontal slicing of the murine brain, and discuss challenges and potential pitfalls of this technique. Finally, we will show some examples for the use of brain slices in further applications.

This article aims to describe a systematic protocol to obtain horizontal hippocampal brain slices in mice. The objective of this methodology is to preserve the integrity of hippocampal fiber pathways, such as the perforant path and the mossy fiber tract to assess dentate gyrus related neurological processes.

Rebanadas hipocampales horizontales del cerebro del ratón

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

Imágenes transpupilares de dos fotones in vivo de la retina del ratón

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...