Los autores agradecen la asistencia de la señora Sharon Spana en el monitoreo de roedores, la manipulación y la experimentación. Apoyo financiero de doctorado ha sido proporcionada por la Universidad de Sydney y el Centro Australiano de Investigación de Excelencia en la visión.

Este protocolo sigue las directrices de cuidado de animales de la Universidad de Sydney. 1. Electrodos Fabricadores <ol><li> Construir el electrodo positivo (el que se pondrá en contacto la córnea) a partir de un corto (5 cm) longitud de alambre de platino 1-2 mm de diámetro. Moda en un bucle de unos pocos milímetros de diámetro. Conecte este lazo a una ventaja convencional, el tiempo suficiente para llegar a la etapa de entrada del amplificador (ver Figura 4). </li><li> Construir el electrodo negativo (que irá en la boca del animal) utilizando un Ag / AgCl pellet 1-2 mm de diámetro, también conectado a una ventaja de convención (véase la Figura 4). </li><li> Como electrodo de referencia (que entrará en la grupa del animal), utilice una aguja hipodérmica limpia (23 G), también conectado a un cable de longitud adecuada (ver Figura 4). </li><li> Lo ideal es usar cables de tres líderes proporcionadas por los fabricantes de instrumentos, para conectar los tres electrodos (U positivo94; córnea, negativo → boca, la referencia → grupa) al amplificador. </li></ol> 2. Conexión y calibración del estímulo de luz y ERG Set-up <ol><li> Crear (o localizar a) un laboratorio de grabación pequeño, que puede hacerse oscuro. Equipar con uno o ambos de una luz sobre-el-banco hecho de color rojo o una cabeza con lámpara roja. </li><li> Use un medidor de luz para confirmar que la iluminación de luz roja alcanzando el ojo de la rata durante la instalación no exceda de 1 lux. Nota: un filtro de densidad neutra se puede utilizar para reducir el brillo de la lámpara y la fuente de luz de la lámpara debe emitir específicamente luz roja. Adaptación oscuro se verá comprometida si las fuentes de luz emiten bajas (longitudes de onda visibles). </li><li> Sellar toda la luz parásita que entra en el laboratorio de grabación (esto a menudo requiere persistencia con cinta opaca) y preparar un filtro de densidad neutra (esto se puede comprar en las hojas) lo suficientemente grande como para caber en, y tan tenue, cualquier pantalla de ordenador que tendrá en el laboratorio. Nota: La luz parásita yla luz de una pantalla son suficientes para perjudicar adaptación a la oscuridad de los ojos de rata. </li><li> Conecte el amplificador al hardware de adquisición de datos. Conecte cables positivo, negativo y de referencia para el amplificador. Asegúrese de que el ordenador y la fuente de alimentación Ganzfeld LED estén bien conectados a una fuente de tierra. Nota: Algunos laboratorios han especializado puntos de puesta a tierra, conectado a una tierra del edificio; una pipa de agua es una alternativa eficaz. </li><li> Calibrar la fuente de luz LED con un radiómetro-calidad de la investigación. Fije el sensor del medidor en la posición en la que los ojos del animal se encuentra durante un experimento. </li><li> Programa de los LED Ganzfeld para ejecutar un protocolo ERG de campo completo con aumentos graduales en la energía flash, duración del flash, flash de repetición y el tiempo entre destellos, denominado intervalo interstimuls (ISI), los ajustes. Por ejemplo, un protocolo de campo completo véase el cuadro 1. Nota: El campo completo ERG destellos aumentan de destellos tenues repetitivas abderecha parpadea de una manera sabia paso. El programa de flash doble continuación del protocolo de campo completo y permite el aislamiento de las respuestas de conos y bastones. </li></ol> 3. Día Antes de ERG Experimentación <ol><li> Oscuro adaptarse ratas Sprague-Dawley de 12 hr antes de la grabación. Es conveniente hacer esto en el laboratorio de grabación, una vez que la luz externa se ha eliminado. </li></ol> 4. Día de ERG Experimentación <ol><li> Haga arreglos para que el animal sea calienta suavemente durante la grabación. Utilizamos una plataforma de metal ligero construido de manera que la cabeza del animal puede descansar en el punto correcto en la entrada a la Ganzfeld. La plataforma tiene tubo incorporado a través del cual bombeamos agua precalentada a 40 ° C en un baño de agua. Nota: La experiencia demuestra que esto mantiene la temperatura central del animal a 37 ° C. </li><li> Pesar la rata en condiciones de oscuridad. Apunte el peso y compensar ketamina correcta (60 mg / kg) y xilazina (5 mg / kg) dosis. Restringir la rata gently e inyectar anestesia por vía intraperitoneal. </li><li> Nota momento de la inyección. Una vez que el animal está inconsciente (generalmente dentro de 5 min) comprobar la profundidad de la anestesia ligeramente pellizcar una almohadilla del pie, para ver si una respuesta refleja está presente. Lo mejor es esperar hasta que este reflejo está ausente o débil, antes de proceder. </li><li> Aplique una gota de atropina y otro de proxmethacaine a la córnea. </li><li> Cortar una longitud de 10 cm de hilo negro. Haga un lazo con un nudo simple y deslice el aro sobre el ecuador del ojo. Apriete ligeramente; el efecto es dibujar el globo ocular ligeramente hacia adelante, con una presión mínima. Esto mantiene la córnea clara de los párpados. </li><li> Aplicar ojo carbómero cae a la superficie de la córnea. Asegurar carbómero permanece en la superficie de la córnea y no se derrame en los párpados o la cara. </li><li> Coloque la ropa de cama absorbente en la parte superior de la plataforma climatizada. </li><li> Posición de la rata en la ropa de cama, con la cabeza en el lugar recomendado en la apertura de la Ganzfeld. </li><li> Insertar intsonda de temperatura ernal en el recto. Sonda de temperatura segura en posición con cinta adhesiva el cable de la sonda a la cola. </li><li> Inserte el electrodo de referencia (la aguja 23 G) por vía subcutánea en la pata trasera, y conectarse a un amplificador. </li><li> Coloque el electrodo negativo (el sedimento de Ag / AgCl) de forma segura en la boca. Para evitar que esto se salga de la boca, colocar el cable de conexión a una superficie estable. </li><li> Coloque el electrodo positivo sobre el centro de la córnea. El uso de un micromanipulador, asegúrese de que el electrodo toca la córnea con suavidad. </li><li> Revise la temperatura del cuerpo está en 37,0 a 37,5 ° C. </li><li> Una vez que el animal esté correctamente colocado y electrodos están en su lugar, cubrir toda la configuración (Ganzfeld y animal) con un material opaco (para preservar la adaptación a la oscuridad). Utilizamos un paño negro suave. </li><li> En el software de adquisición ajustado a una frecuencia de muestreo de 2 KHz con un tiempo de colección de 100 a 1000 ms con 5 ms de muestreo pre-colección. Establezca los filtros de paso de banda para 1-1.000Hz y asegurar que el muestreo se activa para probar el periodo de ~ 250 ms después de un flash. </li><li> Compruebe la línea de base de la grabación. Debe estar libre de ruidos extraños, pero muestran un poco de ruido del amplificador y una oscilación respiratoria. </li><li> Si la línea de base muestra ruidos extraños, comenzará la solución de problemas. La mayoría de los problemas están relacionados con el deslizamiento en la posición del electrodo, o de puesta a tierra. Utilice una jaula de Faraday para garantizar grabaciones están libres de ruidos extraños. </li><li> Ejecute un destello de prueba, 0,4 log scot cd.sm -2. Una forma de onda ERG similar a la figura debería aparecer 2A. En nuestras respuestas típicas de laboratorio para un 0,4 log scot cd.sm -2 Flash son (una onda: -474 ± 39 mV y la onda b: 1.512 ± 160 mV, n = 11). </li><li> Deje que los animales a oscuro re-adaptarse durante 10 min. Es conveniente utilizar estos 10 min para volver a comprobar la línea de base. </li><li> Tras la confirmación de señal estable comenzar la grabación. </li><li> Al final de la sesión de grabación, compruebe que temperat cuerpose mantuvo ure. Retire los electrodos. Vuelva a aplicar polímero carbómero de córneas. Deje que el animal se recupere en una almohadilla de calor hasta que es completamente móvil y activo, antes de regresar a la vivienda de los animales. </li></ol> 5. La isquemia a distancia <ol><li> Realizar isquemia a distancia, ya sea en roedores despiertos o anestesiados. </li><li> Si se anestesia el animal, lo pondré sobre una plataforma climatizada (arriba) y deslice el manguito del esfigmomanómetro sobre la parte superior de la extremidad posterior, clara de la rodilla. </li><li> Si los animales están acostumbrados a ser manejado, es posible llevar a cabo este procedimiento sin anestesia; esto requiere dos personas. A una persona le restringe el animal suavemente, y la segunda se aplica el manguito del esfigmomanómetro y opera el esfigmomanómetro. </li><li> Para los animales despiertos, use un pedazo de toalla de ~ 15 cm x 30 a 50 cm para envolver suavemente el animal, con un solo miembro posterior libre. Colocar el animal en la espalda a (por ejemplo) el antebrazo izquierdo, con la cabeza metida entre el brazo y el torso del titular, y el lugarel brazalete como se acaba de describir. </li><li> Desinflar el manguito y asegurar la válvula de presión de aire se cierra. Bombee el manguito a 160 mmHg en animales anestesiados, y 180 mmHg en animales despiertos. Esto supera la presión sistólica (generalmente 140 mmHg y 160 mmHg respectivamente). </li><li> Mantener estas presiones, según sea necesario, utilizando la bomba de mano. </li><li> Después de la hora prevista para la isquemia (usamos 2 períodos de 5 min separadas por 5 min de reperfusión), desinflar la presión del manguito aflojando la válvula de presión de aire. </li><li> Confirmar el efecto de la isquemia a distancia con una sonda de temperatura de la piel unida a la almohadilla de la pata. Temperatura de la piel por lo general cae 32-30 ° C, durante 5 min y se recupera en la reperfusión. </li></ol> 6. Daño Luz <ol><li> Asegúrese de que las ratas están en un oscuro adaptado durante la noche, antes de que el procedimiento de daño de la luz. </li><li> En el momento apropiado después de isquemia de las extremidades (en nuestros experimentos sin demora), cada animal se coloca en solitario en una cajas de plexiglás, wiª de agua y alimentos en recipientes con base de suelo. Nota: El daño inducido por la luz sólo puede llevarse a cabo en animales albinos. </li><li> Encender una luz blanca pre-calibrado 1.000 lux a un tiempo estándar (normalmente 09 a.m.) y mantener esta condición durante 24 horas. </li></ol> 7. ERG extracción de datos y análisis <ol><li> Adquirir formas de onda promediados del ERG. Si es necesario, correcto para una línea de base distinto de cero, por sustracción. </li><li> Medir la amplitud de la onda a (presentado en mediados a altas intensidades de estímulo), como la diferencia de voltaje entre la línea de base y la primera (&lt;30 mseg latencia) canal (Figura 1). </li><li> Mida la amplitud de la onda b como la diferencia de voltaje entre el pico de la onda A y el positivo de la siguiente ola, que se producen normalmente en una latencia de 80 a 100 ms (Figura 1). </li><li> Aislar potenciales oscilatorios mediante el uso de una transformada de Fourier para filtrar los datos 60 a 235 Hz, con una banda de transición 90 Hz <sup&gt; 7. Si se requiere la señal potencial oscilatorio aislado puede entonces ser restada de la forma de onda sin filtrar para confirmar la identidad de la vaguada de onda a. </li><li> El tiempo implícito (latencia) de los picos A y B de onda también puede ser una medida útil (Figura 1). Utilice los destellos individuales para aislar la respuesta varilla. Restar la respuesta de los conos (flash 2) de la respuesta mixta (flash 1) para aislar la respuesta de varilla (Figura 2). </li><li> Normalizar la intensidad de luz individual una onda y amplitudes de onda b (post-tratamiento / post-tratamiento-base) o un promedio de grupos de tratamiento. Curvas Intensidad-respuesta trazar las amplitudes de grupo y de error contra la energía flash. </li></ol>

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Investigative Ophthalmology &amp; Visual Science. 55 (4), 2088-2096 (2014).</li><li>Maccarone, R., Di Marco, S., Bisti, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Saffron+supplement+maintains+morphology+and+function+after+exposure+to+damaging+light+in+mammalian+retina.">Saffron supplement maintains morphology and function after exposure to damaging light in mammalian retina.</a> Investigative Ophthalmology &amp; Visual Science. 49 (3), 1254-1261 (2008).</li><li>Hood, D. C., Birch, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+abnormal+rod+photoreceptor+activity+with+the+a-wave+of+the+electroretinogram:+Applications+and+methods.">Assessing abnormal rod photoreceptor activity with the a-wave of the electroretinogram: Applications and methods.</a> Documenta Ophthalmologica. 92 (4), 253-267 (1996).</li><li>Robson, J. G., Frishman, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+rod-driven+a-wave+of+the+dark-adapted+mammalian+electroretinogram.">The rod-driven a-wave of the dark-adapted mammalian electroretinogram.</a> Progress in Retinal and Eye Research. 39, 1-22 (2014).</li><li>Hood, D. C., Birch, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+COMPUTATIONAL+MODEL+OF+THE+AMPLITUDE+AND+IMPLICIT+TIME+OF+THE+B-WAVE+OF+THE+HUMAN+ERG.">A COMPUTATIONAL MODEL OF THE AMPLITUDE AND IMPLICIT TIME OF THE B-WAVE OF THE HUMAN ERG.</a> Visual Neuroscience. 8 (2), 107-126 (1992).</li><li>Wachtmeister, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oscillatory+potentials+in+the+retina:+what+do+they+reveal.">Oscillatory potentials in the retina: what do they reveal.</a> Progress in Retinal and Eye Research. 17 (4), 485-521 (1998).</li></ol>

Jonathan Stone es el director de CSCM Pty Ltd

El método ERG flash adaptada a la oscuridad descrito anteriormente es un método fiable para evaluar la función de la retina en ratas. Tanto la onda ay la onda b se redujeron en un daño de la luz. Remoto precondicionamiento isquémico mitigado reducciones inducidas daños-luz en la onda ay la onda b. Esta preservación de la función retiniana sugiere que el precondicionamiento isquémico remoto ha inducido neuroprotección, asemejándose a otras formas de acondicionamiento previo de protección, tales como hipoxia, ...

El ERG es un potencial eléctrico generado por la retina en respuesta a la luz, y grabado de la superficie corneal del ojo. Cuando las condiciones de grabación se manejan con cuidado, el ERG se puede utilizar en una variedad de maneras para evaluar la función de la retina. Aquí describimos cómo grabar el &#39;Flash ERG&#39;, el potencial generado cuando la retina se expone a un breve, flash brillante presentado en un fondo Ganzfeld. El Ganzfeld dispersa la luz de forma homogénea y el flash de la luz alcanza toda la retina aproximadamente uniforme. Si la retina está oscuro adaptado antes de grabar, y la oscuridad, la adaptación se mantiene como el animal se prepara para la grabación, el ERG obtenido se genera por ambas fotorreceptores conos y bastones. El oscuro-adaptado de flash ERG tiene una forma de onda característica, que ha sido analizada de dos maneras. En primer lugar, principios y finales de los componentes de la forma de onda ERG se han distinguido, y en relación con la secuencia de la neuronaal activación en la retina. El primer componente es una latencia corta en sentido negativo potencial, la onda a (Figura 1). Esto es seguido por un potencial positivo va, llamada la onda b. La fase ascendente de la onda b muestra oscilaciones, que se consideran un componente separado (potenciales oscilatorios o PO). La onda a se considera que está generada por fotorreceptores, la onda b por las células de la capa nuclear interna, y los PO por las células amacrinas 1. Sobre la base de la fuerza del estímulo, las respuestas a destellos muy tenues denominan el umbral de respuesta escotópica son posibles. El umbral de respuesta escotópica se entiende que ser generado a partir de las células ganglionares de la retina 2-4. En segundo lugar, el flash ERG se puede separar por adaptación a la luz, o por un protocolo de dos flash descritos a continuación, en componentes de bastón y cono impulsada. En condiciones fotópicas, la onda a es no detectable en ratas, porque la población de cono es baja, pero PO y una onda b sonclaro 5. En los primates, cuyas retinas tienen poblaciones cono superior, tanto de varilla y las vías de cono generan una detectable una onda 6. Dos medidas útiles a menudo extraídos de la ERG flash son las amplitudes de la a- y b-ondas, medidos como en la Figura 1, con respuestas de flash típicos mostrados en la Figura 2. Cuando la población fotorreceptor se reduce, por ejemplo por la exposición a damagingly brillante luz, se reducen todos los componentes del ERG. Intervenciones neuroprotectoras, como a distancia precondicionamiento isquémico (RIP), pueden ser validados por la preservación de las amplitudes de la a- y b-ondas (Figura 3). En resumen, el análisis de la ERG permite comparaciones entre sana, ligera y de la retina dañada neuroprotected.

<table border="0" cellpadding="0" cellspacing="0">
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		<tr>
			<td>PC computer</td>
			<td></td>
			<td></td>
			<td></td>
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		<tr>
			<td>Powerlab, 4 channel acquistion hardware</td>
			<td>AD Instruments</td>
			<td>PL 35044</td>
			<td>Acquistion of ERG</td>
		</tr>
		<tr>
			<td>Animal Bio Amp</td>
			<td>AD Instruments</td>
			<td>FE 136</td>
			<td>Amplifier for ERG</td>
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		<tr>
			<td>Lab chart</td>
			<td>AD Instruments</td>
			<td></td>
			<td>Signal collection software</td>
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		<tr>
			<td>Ganzfield</td>
			<td>Photometric solutions</td>
			<td>FS-250A</td>
			<td>Light stimulus</td>
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		<tr>
			<td>Ganzfield operating system</td>
			<td>Photometric solutions</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Research Radiometer</td>
			<td>International light technologies</td>
			<td>ILT-1700</td>
			<td>calibrate light series</td>
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		<tr>
			<td>Lux meter</td>
			<td></td>
			<td>LX-1010B&#160;</td>
			<td>check red light illumanation</td>
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		<tr>
			<td>Excel</td>
			<td>microsoft</td>
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			<td></td>
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			<td>Lead wires</td>
			<td>AD Instruments</td>
			<td></td>
			<td>Connect postive, negative ground electrodes to amplifier</td>
		</tr>
		<tr>
			<td>Lead wires -aligator</td>
			<td>AD Instruments</td>
			<td></td>
			<td>ground ganzfield and acquistion hardware to computer</td>
		</tr>
		<tr>
			<td>Platinum wire 95%</td>
			<td>A&#38;E metals</td>
			<td></td>
			<td>postive electrode</td>
		</tr>
		<tr>
			<td>Mouth electrode Ag/AgCl Pellet</td>
			<td>SDR</td>
			<td>E205</td>
			<td>negative electode</td>
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			<td>26 gauge needle</td>
			<td>BD</td>
			<td></td>
			<td>ground electode</td>
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			<td>Water pump</td>
			<td></td>
			<td></td>
			<td></td>
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		<tr>
			<td>Water bath</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tubing</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Homeothermic blanket system with flexible probe</td>
			<td>Harvard Appartus</td>
			<td>507222F</td>
			<td></td>
		</tr>
		<tr>
			<td>Atropine 1% w/v</td>
			<td>Bausch &#38; Lomb</td>
			<td></td>
			<td>topical mydriasis</td>
		</tr>
		<tr>
			<td>Proxmethycaine 0.5% w/v</td>
			<td>Bausch &#38; Lomb</td>
			<td></td>
			<td>topical anaesthetic</td>
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		<tr>
			<td>Visco tears eye drops</td>
			<td>Novartis</td>
			<td></td>
			<td>carbomer polymer</td>
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			<td>Thread</td>
			<td></td>
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			<td>retract eye lid</td>
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			<td>Tweezers</td>
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			<td>Reusable adhesive</td>
			<td>Blu tac</td>
			<td></td>
			<td>Dim red headlamp. Affix electrodes</td>
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			<td>Absorbent bedding</td>
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			<td>Ketamil - ketamine 100 mg/ml - 50 ml</td>
			<td>Troy Laboratories Pty Ltd</td>
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			<td>dissociative</td>
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			<td>Xylium - Xylazine 100 mg/ml - 50 ml</td>
			<td>Troy Laboratories Pty Ltd</td>
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			<td>muscle relaxant</td>
		</tr>
		<tr>
			<td>Scale</td>
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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.

El protocolo se puede utilizar para medir la función visual de la retina de roedores in vivo. La onda a, una medida de la función de los fotorreceptores, y la onda b, una medida de la función de la retina interna, se anotan en la Figura 1. Las señales ERG aumentos de barra dominadas con el creciente estímulo de luz, como se muestra en la Figura 2A. La onda se convierte en un aparente en ~ 0,4 log scot cd.sm -2 y la amplitud de la ond...

Watch this Scientific Journal Video about Using the Electroretinogram to Assess Function in the Rodent Retina and the Protective Effects of Remote Limb Ischemic Preconditioning at JoVE.com

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

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 ...

using-electroretinogram-to-assess-function-rodent-retina-protective

Research

JoVE Journal

Neuroscience

16.0K vistas.  University of Sydney. 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.

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

Strategies for Study of Neuroprotection from Cold-preconditioning

Neurological injury is a frequent cause of morbidity and mortality from general anesthesia and related surgical procedures that could be alleviated by development of effective, easy to administer and safe preconditioning treatments. We seek to define the neural immune signaling responsible for cold-preconditioning as means to identify novel targets for therapeutics development to protect brain before injury onset. Low-level pro-inflammatory mediator signaling changes over time are essential for cold-preconditioning neuroprotection. This signaling is consistent with the basic tenets of physiological conditioning hormesis, which require that irritative stimuli reach a threshold magnitude with sufficient time for adaptation to the stimuli for protection to become evident.
Accordingly, delineation of the immune signaling involved in cold-preconditioning neuroprotection requires that biological systems and experimental manipulations plus technical capacities are highly reproducible and sensitive. Our approach is to use hippocampal slice cultures as an in vitro model that closely reflects their in vivo counterparts with multi-synaptic neural networks influenced by mature and quiescent macroglia / microglia. This glial state is particularly important for microglia since they are the principal source of cytokines, which are operative in the femtomolar range. Also, slice cultures can be maintained in vitro for several weeks, which is sufficient time to evoke activating stimuli and assess adaptive responses. Finally, environmental conditions can be accurately controlled using slice cultures so that cytokine signaling of cold-preconditioning can be measured, mimicked, and modulated to dissect the critical node aspects. Cytokine signaling system analyses require the use of sensitive and reproducible multiplexed techniques. We use quantitative PCR for TNF-&alpha; to screen for microglial activation followed by quantitative real-time qPCR array screening to assess tissue-wide cytokine changes. The latter is a most sensitive and reproducible means to measure multiple cytokine system signaling changes simultaneously. Significant changes are confirmed with targeted qPCR and then protein detection. We probe for tissue-based cytokine protein changes using multiplexed microsphere flow cytometric assays using Luminex technology. Cell-specific cytokine production is determined with double-label immunohistochemistry. Taken together, this brain tissue preparation and style of use, coupled to the suggested investigative strategies, may be an optimal approach for identifying potential targets for the development of novel therapeutics that could mimic the advantages of cold-preconditioning.

We seek to define the neural immune signaling responsible for cold-preconditioning as means to identify novel targets for therapeutics development to protect brain before injury onset. We present strategies for such work that require biological systems, experimental manipulations plus technical capacities that are highly reproducible and sensitive.

Estrategias para el Estudio de la neuroprotección de frío preacondicionamiento

Neurological injury is a frequent cause of morbidity and mortality from general anesthesia and related surgical procedures that could be alleviated by ...

Investigación

Neurociencias

Tissue Preparation and Immunostaining of Mouse Sensory Nerve Fibers Innervating Skin and Limb Bones

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in animals and humans. These peripheral sensory nerve fibers express a plethora of receptors and ion channel proteins which detect and initiate specific sensory stimuli. Methods are available to characterize the electrical properties of peripheral sensory nerve fibers innervating the skin, which can also be utilized to identify the functional expression of specific ion channel proteins in these fibers. However, similar electrophysiological methods are not available (and are also difficult to develop) for the detection of the functional expression of receptors and ion channel proteins in peripheral sensory nerve fibers innervating other visceral organs, including the most challenging tissues such as bone. Moreover, such electrophysiological methods cannot be utilized to determine the expression of non-excitable proteins in peripheral sensory nerve fibers. Therefore, immunostaining of peripheral/visceral tissue samples for sensory nerve fivers provides the best possible way to determine the expression of specific proteins of interest in these nerve fibers. So far, most of the protein expression studies in sensory neurons have utilized immunostaining procedures in sensory ganglia, where the information is limited to the expression of specific proteins in the cell body of specific types or subsets of sensory neurons. Here we report detailed methods/protocols for the preparation of peripheral/visceral tissue samples for immunostaining of peripheral sensory nerve fibers. We specifically detail methods for the preparation of skin or plantar punch biopsy and bone (femur) sections from mice for immunostaining of peripheral sensory nerve fibers. These methods are not only key to the qualitative determination of protein expression in peripheral sensory neurons, but also provide a quantitative assay method for determining changes in protein expression levels in specific types or subsets of sensory fibers, as well as for determining the morphological and/or anatomical changes in the number and density of sensory fibers during various pathological states. Further, these methods are not confined to the staining of only sensory nerve fibers, but can also be used for staining any types of nerve fibers in the skin, bones and other visceral tissue.

Immunocytochemical identification of peripheral sensory nerve fiber subtypes (and detection of protein expression therein) are key to the understanding of molecular mechanisms underlying peripheral sensation. Here we describe methods for preparation of peripheral/visceral tissue samples, such as skin and limb bones, for specific immunostaining of peripheral sensory nerve fibers.

Preparación y tejido de fibras inmunotinción ratón nerviosas sensoriales que inervan la piel y los huesos de extremidades

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in ...

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

Entrega intraventricular directa de medicamentos al roedor Sistema Nervioso Central

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). It is increasingly being recognized that several neurodegenerative diseases such as Alzheimer&#8217;s, multiple sclerosis, and amyotrophic lateral sclerosis present elements of vascular compromise. In addition, the most prominent causes of blindness in pediatric and working age populations (retinopathy of prematurity and diabetic retinopathy, respectively) are characterized by vascular degeneration and failure of physiological vascular regrowth. The aim of this technical paper is to provide a detailed protocol to study CNS vascular regeneration in the retina. The method can be employed to elucidate molecular mechanisms that lead to failure of vascular growth after ischemic injury. In addition, potential therapeutic modalities to accelerate and restore healthy vascular plexuses can be explored. Findings obtained using the described approach may provide therapeutic avenues for ischemic retinopathies such as that of diabetes or prematurity and possibly benefit other vascular disorders of the CNS.

The rodent retina has long been recognized as an accessible window to the brain. In this technical paper we provide a protocol that employs the mouse model of oxygen-induced retinopathy to study the mechanisms that lead to failure of vascular regeneration within the central nervous system after ischemic injury. The described system can also be harnessed to explore strategies to promote regrowth of functional blood vessels within the retina and CNS.

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). ...

Electroretinogram Analysis of the Visual Response in Zebrafish Larvae

The electroretinogram (ERG) is a noninvasive electrophysiological method for determining retinal function. Through the placement of an electrode on the surface of the cornea, electrical activity generated in response to light can be measured and used to assess the activity of retinal cells in vivo. This manuscript describes the use of the ERG to measure visual function in zebrafish. Zebrafish have long been utilized as a model for vertebrate development due to the ease of gene suppression by morpholino oligonucleotides and pharmacological manipulation. At 5-10 dpf, only cones are functional in the larval retina. Therefore, the zebrafish, unlike other animals, is a powerful model system for the study of cone visual function in vivo. This protocol uses standard anesthesia, micromanipulation and stereomicroscopy protocols that are common in laboratories that perform zebrafish research. The outlined methods make use of standard electrophysiology equipment and a low light camera to guide the placement of the recording microelectrode onto the larval cornea. Finally, we demonstrate how a commercially available ERG stimulator/recorder originally designed for use with mice can easily be adapted for use with zebrafish. ERG of larval zebrafish provides an excellent method of assaying cone visual function in animals that have been modified by morpholino oligonucleotide injection as well as newer genome engineering techniques such as Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9, all of which have greatly increased the efficiency and efficacy of gene targeting in zebrafish. In addition, we take advantage of the ability of pharmacological agents to penetrate zebrafish larvae to evaluate the molecular components that contribute to the photoresponse. This protocol outlines a setup that can be modified and used by researchers with various experimental goals.

We present a method for the electroretinographic (ERG) analysis of zebrafish larvae utilizing micromanipulation and electroretinography techniques. This is a simple and straightforward method for assaying visual function of zebrafish larvae in vivo.

Análisis Electrorretinograma de la respuesta visual en pez cebra larvas

The electroretinogram (ERG) is a noninvasive electrophysiological method for determining retinal function. Through the placement of an electrode on the ...

Imaging Serotonergic Fibers in the Mouse Spinal Cord Using the CLARITY/CUBIC Technique

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal cord of the majority of these fiber systems have not been thoroughly investigated in any species. Serotonergic fibers, which project to the spinal cord, have been studied in rats and opossums on histological sections and their functional significance has been deduced based on their fiber termination pattern in the spinal cord. With the development of CLARITY and CUBIC techniques, it is possible to investigate this fiber system and its distribution in the spinal cord, which is likely to reveal previously unknown features of serotonergic supraspinal pathways. Here, we provide a detailed protocol for imaging the serotonergic fibers in the mouse spinal cord using the combined CLARITY and CUBIC techniques. The method involves perfusion of a mouse with a hydrogel solution and clarification of the tissue with a combination of clearing reagents. Spinal cord tissue was cleared in just under two weeks, and the subsequent immunofluorescent staining against serotonin was completed in less than ten days. With a multi-photon fluorescent microscope, the tissue was scanned and a 3D image was reconstructed using Osirix software.

Supraspinal projections are important for pain perception and other behaviors, and serotonergic fibers are one of these fiber systems. The present study focused on the application of the combined CLARITY/CUBIC protocol to the mouse spinal cord in order to investigate the termination of these serotonergic fibers.

Las fibras serotoninérgicos proyección de imagen en la médula espinal de ratón El uso de la claridad / Técnica CÚBICO

Long descending fibers to the spinal cord are essential for locomotion, pain perception, and other behaviors. The fiber termination pattern in the spinal ...

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous system. SC-selective genetic manipulation in living animals is a powerful technique for studying the molecular and cellular mechanisms of SC myelination and demyelination in vivo. While knockout/knockin and transgenic mice are powerful tools for studying SC biology, these methods are costly and time consuming. Viral vector-mediated transgene introduction into the sciatic nerve is a simpler and less laborious method. However, viral methods have limitations, such as toxicity, transgene size constraints, and infectivity restricted to certain developmental stages. Here, we describe a new method that allows selective transfection of myelinating SCs in the rodent sciatic nerve using electroporation. By applying electric pulses to the sciatic nerve at the site of plasmid DNA injection, genes of interest can be easily silenced or overexpressed in SCs in both neonatal and more mature animals. Furthermore, this in vivo electroporation method allows for highly efficient simultaneous expression of multiple transgenes. Our novel technique should enable researchers to efficiently manipulate SC gene expression, and facilitate studies on SC development and function.

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs.

En Vivo Transferencia de genes a células de Schwann en el nervio ciático de roedores mediante electroporación

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous ...

Effects of Blast-induced Neurotrauma on Pressurized Rodent Middle Cerebral Arteries

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral vascular effects. Impact (i.e., non-blast) traumatic brain injury (TBI) is known to decrease pressure autoregulation in the cerebral vasculature in both humans and experimental animals. The hypothesis that blast-induced traumatic brain injury (bTBI), like impact TBI, results in impaired cerebral vascular reactivity was tested by measuring myogenic dilatory responses to reduced intravascular pressure in rodent middle cerebral arterial (MCA) segments from rats subjected to mild bTBI using an Advanced Blast Simulator (ABS) shock tube. Adult, male Sprague-Dawley rats were anesthetized, intubated, ventilated and prepared for Sham bTBI (identical manipulation and anesthesia except for blast injury) or mild bTBI. Rats were randomly assigned to receive Sham bTBI or mild bTBI followed by sacrifice 30 or 60 min post-injury. Immediately after bTBI, righting reflex (RR) suppression times were assessed, euthanasia at the time points post-injury was completed, the brain was harvested and the individual MCA segments were collected, mounted and pressurized. As the intraluminal pressure perfused through the arterial segments was reduced in 20 mmHg increments from 100 to 20 mmHg, MCA diameters were measured and recorded. With decreasing intraluminal pressure, MCA diameters steadily increased significantly above baseline in the Sham bTBI groups while MCA dilator responses were significantly reduced (p &#60; 0.05) in both bTBI groups as evidenced by the impaired, smaller MCA diameters recorded for the bTBI groups. In addition, RR suppression in the bTBI groups was significantly (p &#60; 0.05) higher than in the Sham bTBI groups. MCA&#39;s collected from the Sham bTBI groups exhibited typical vasodilatory properties to decreases in intraluminal pressure while MCA&#39;s collected following bTBI exhibited significantly impaired myogenic vasodilatory responses to reduced pressure that persisted for at least 60 min after bTBI.

Here, we present a protocol to describe methods for ex vivo vascular reactivity determination following a primary blast traumatic brain injury (bTBI) using isolated, pressurized, rodent middle cerebral arterial (MCA) segments. bTBI induction is accomplished using a shock tube, also known as an Advanced Blast Simulator (ABS) device.

Efectos de Neurotrauma inducido ráfaga en roedor a presión media de las arterias cerebrales

Though there have been studies on the histopathological and behavioral effects of blast exposure, fewer have been dedicated to blast&#39;s cerebral ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

Análisis cinemático 3D para la evaluación funcional en el modelo de rata de lesión de aplastamiento de nervios ciáticos

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...

Combining Behavior and EEG to Study the Effects of Mindfulness Meditation on Episodic Memory

Although there has been recent interest in how mindfulness meditation can affect episodic memory as well as brain structure and function, no study has examined the behavioral and neural effects of mindfulness meditation on episodic memory. Here we present a protocol that combines mindfulness meditation training, an episodic memory task, and EEG to examine how mindfulness meditation changes behavioral performance and the neural correlates of episodic memory. Subjects in a mindfulness meditation experimental group were compared to a waitlist control group. Subjects in the mindfulness meditation experimental group spent four weeks training and practicing mindfulness meditation. Mindfulness was measured before and after training using the Five Facet Mindfulness Questionnaire (FFMQ). Episodic memory was measured before and after training using a source recognition task. During the retrieval phase of the source recognition task, EEG was recorded. The results showed that mindfulness, source recognition behavioral performance, and EEG theta power in right frontal and left parietal channels increased following mindfulness meditation training. In addition, increases in mindfulness correlated with increases in theta power in right frontal channels. Therefore, results obtained from combining mindfulness meditation training, an episodic memory task, and EEG reveal the behavioral and neural effects of mindfulness meditation on episodic memory.

Here we present a protocol for combining mindfulness meditation training, an episodic memory task, and EEG to understand the behavioral and neural effects of mindfulness meditation on episodic memory.

Combinación de comportamiento y EEG para estudiar los efectos de la meditación de atención plena en la memoria episódica

Although there has been recent interest in how mindfulness meditation can affect episodic memory as well as brain structure and function, no study has ...