Reconocemos las siguientes fuentes de financiación: NIH NINDS – K08NS101122 (JB), R01NS040337 (JK), R01NS044370 (JK), Facultad de Medicina de la Universidad de Virginia (JB).

Todos los estudios en animales fueron aprobados por el Comité Institucional de Cuidado y Uso de Animales (IACUC) de la Universidad de Virginia.
1. Edificio de electrodos / edificio de cables
<ol><li>Utilice un cable de acero inoxidable con aislamiento unipolar (diámetro desnudo de 0,005", recubierto de 0,008") para hacer un electrodo que esté conectado con un conector de enchufe hembra (conector de receptáculo hembra 0.079).</li>
	<li>Utilice un cable especial hecho a medida para conectar animales al amplificador.<ol>
<li>Conecte un conector macho de 4 pines (conector macho 0.079") a un amplificador operacional (amplificador operacional) de impedancia de ganancia de unidad de 4 canales. Conecte una resistencia de 10K a los cables que se conectan a la batería de 9 V. Un cable de tierra no conectado al amplificador operacional actúa como el punto medio de la batería.</li>
		<li>Conecte un extremo del cable (AWG, 0.012" OD) al amplificador operacional y conecte el otro extremo del cable al amplificador.</li>
	</ol>
</li>
</ol>2. Cirugía de implantación de electrodos
<ol><li>Anestesiar al cachorro (día postnatal 9) con 4-5% de isoflurano en una capucha de flujo descendente. Antes del inicio del procedimiento, inyecte a los cachorros con bupivacaína (0.02-0.05 mL, infiltración local subcutánea al 0.25%).</li>
	<li>Una vez que el animal está inmóvil, transfiéralo a una etapa estereotáctica con un cono nasal. Use el reverso de la barra de la oreja, ya que es suave para mantener la cabeza firme. A esta edad, los cachorros no tienen una oreja completamente desarrollada para usar el extremo puntiagudo de la barra de la oreja.</li>
	<li>Reduzca el flujo de isoflurano y manténgalo en 2.5-3%. Vigile la respiración constante del cachorro durante todo el procedimiento quirúrgico. Pellizque la cola para verificar la respuesta al dolor y luego proceda a la incisión.</li>
	<li>Esterilizar el área de incisión en el cráneo con betadina y alcohol (3 ciclos de yodo alterno y etanol al 70%). Cubra la parte circundante del cuerpo de tal manera que la región de la incisión sea visible.</li>
	<li>Abra el cuero cabelludo anterior-posterior desde ligeramente por encima de los ojos y retraiga aproximadamente 0,5 cm de piel. Reposicione la cabeza del ratón en la etapa estereotáxica para que la piel tire hacia afuera exponiendo el cráneo.</li>
	<li>Aplique peróxido de hidrógeno en el cráneo con un hisopo de algodón y raspe el cráneo limpio con una cuchilla de bisturí. El cráneo es muy suave; tenga cuidado al raspar.</li>
	<li>Aplique una gota (aproximadamente 50 μL) de adhesivo y extiéndalo alrededor del área expuesta del cráneo usando su aplicador. Exponer a la luz UV durante 40 s para fijar el adhesivo.</li>
	<li>Mida las coordenadas utilizando el bregma expuesto como referencia. Implantar electrodos bilateralmente en la región CA1 del hipocampo [-3,5 mm Dorsal-Ventral (DV), ±2 mm Medial-Lateral (ML), -1,75 mm Profundo (D)] y bilateralmente en la corteza parietal [-1,22 mm DV, ±0,5 mm ML, -1 mm D] y un electrodo de referencia en el cerebelo17. Use una aguja de 32 G para crear un agujero en la región marcada.</li>
	<li>Limpie la sangre de la superficie del cráneo. Electrodos inferiores unidos al conector de la cavidad femenina en el cerebro con la ayuda del brazo estereotáxico y fijarlos en su lugar con acrílico dental. Implantar el electrodo en el cerebro. El auricular conector de enchufe se encuentra en la parte superior del cráneo pegado por acrílico dental.</li>
	<li>Inyecte ketoprofeno (5 mg/kg) por vía subcutánea en la región interescapular una vez fijado el electrodo. Coloque a los cachorros de nuevo con la madre. NOTA: Presente la mitad de la camada con los auriculares a la vez a la madre en lugar de presentarlos uno a la vez. Esto evitará que la madre dañe los auriculares del cachorro.</li>
</ol>3. Configuración y registro del EEG (línea de base/pre-lesión)
<ol><li>Después de 24 h de recuperación después de la implantación del electrodo, coloque a cada animal en una cámara de plexiglás calentada (37 ° C) hecha a medida para el registro de EEG. Esta cámara también servirá como una cámara de hipoxia.</li>
	<li>Conecte a los cachorros en la cámara a un sistema de monitoreo de video-EEG a través de un cable flexible (cable op-amp hecho a medida). NOTA: Con los auriculares en su lugar, los ratones son libremente móviles y no exhiben ninguna diferencia en el comportamiento. Una vez conectados a los cables de los electrodos, los cables deben ajustarse dentro de la correa de la cámara para proporcionar la cantidad correcta de holgura para que el cachorro pueda moverse libremente por toda la cámara.</li>
	<li>Digitalice los datos de EEG a 1000 Hz con ganancia de 1K utilizando un amplificador de hierba. Revise la señal EEG (filtro de paso de banda entre 3-70 Hz) más tarde utilizando software (por ejemplo, LabChart Pro).</li>
	<li>Registre un EEG basal previo a la lesión durante 30 minutos antes de desconectar a los animales para el procedimiento de ligadura de la arteria carótida.</li>
</ol>4. Ligadura de la arteria carótida izquierda
<ol><li>Anestesiar al cachorro (día postnatal 10) con 4-5% de isoflurano en una capucha de flujo descendente y colocarlos en una configuración especialmente dispuesta en una almohadilla de baño de agua. Coloque el animal en decúbito supino y asegure las extremidades anteriores con cinta de papel.<ol>
<li>Reducir el flujo de isoflurano a 2-3%. Pellizque la cola para la respuesta al dolor y controle la respiración durante todo el procedimiento.</li>
	</ol>
</li>
	<li>Esterilizar el área de incisión (entre la mandíbula y la clavícula) en el lado izquierdo del cuello con betadina y alcohol (3 ciclos de yodo alterno y etanol al 70%).</li>
	<li>Haga una incisión de aproximadamente 1 cm de largo en el lado izquierdo del cuello usando microescisores. Usando un microscopio de disección, retraiga cuidadosamente el tejido subcutáneo y la piel para exponer la arteria carótida. Tenga cuidado de identificar el nervio vago (que corre lateral a la arteria) y sepárelo y retraiga delicadamente de la arteria.</li>
	<li>Enhebrar una sutura de seda estéril de 5 cm de largo debajo de la arteria usando microfuerzas. Ata una sutura de doble anudado alrededor de la arteria para ocluir el flujo.</li>
	<li>Corte el exceso de sutura y cierre la arteria expuesta tirando hacia atrás del tejido subcutáneo y la piel. Use la unión veterinaria para sellar la incisión.</li>
	<li>Coloque al animal de nuevo en monitoreo continuo de EEG en una cámara a temperatura ambiente, que se coloca en un colchón que se calienta. Realice controles de temperatura infrarrojos puntuales de la temperatura del núcleo del cachorro para evitar abrir la cámara. Permita que el animal se recupere durante 1 h antes de la hipoxia.</li>
</ol>5. EEG e hipoxia
<ol><li>Monitoree continuamente FiO2 (fracción de oxígeno inspirado) dentro de la cámara a través de un monitor de oxígeno.</li>
	<li>Enjuague la cámara con 60 L/min de 100% N2 y 0.415 L/min para 100% de O2. Una vez que la saturación de oxígeno en la cámara alcanza el 12%, disminuya el flujo de N2 a 10 L / min mientras mantiene el flujo de O2 sin cambios. Con pequeños ajustes, mantenga el FiO2 al 8% durante 45 min.</li>
	<li>Después de 45 min de exposición a la hipoxia, devuelva la FiO2 al 21%.</li>
	<li>Haga que los cachorros se recuperen en la cámara y monitoree el EEG durante 2 h después de la hipoxia.</li>
	<li>Después de completar el período de grabación, desconecte los ratones de la grabación de EEG y regrese a la madre.</li>
</ol>6. Análisis EEG
<ol><li>Analice el archivo EEG con video en LabChart Pro. Haga que un investigador ciego marque el EEG para detectar convulsiones y patrones de fondo17. Las convulsiones se definen como un evento electrográfico de duración superior a 10 segundos con descargas rítmicas de onda aguda de alta frecuencia (≥3x basal) con clara evolución17.</li>
	<li>Haga que un segundo investigador ciego revise los eventos marcados al azar para el acuerdo.</li>
	<li>Revisar el video asociado para cada evento electrográfico marcado y analizar de acuerdo con la puntuación de convulsión conductual del roedor neonatal16. Brevemente, esta puntuación oscila entre 0-6 (inmovilidad a comportamiento tónico-clónico severo). Para caracterizar aún más la semiología convulsiva, analice el comportamiento para la lateralidad (movimientos multifocales / bilaterales vs. focales / unilaterales vs. mixtos).</li>
	<li>Cree un espectrograma de potencia. Utilice una transformada rápida de Fourier con una ventana de datos de Cosine-Bell con un tamaño de 1024 puntos de datos. Cree un eje X suave en el espectrograma con la ayuda de una superposición de ventanas del 87,5%. Exprese la potencia como μV218.</li>
</ol>

<ol>
	<li>Vasudevan, C., Levene, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+and+aetiology+of+neonatal+seizures.">Epidemiology and aetiology of neonatal seizures.</a> Seminars in Fetal &amp; Neonatal Medicine. , (2013).</li><li>Volpe, 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=Neonatal+Seizures.">Neonatal Seizures.</a> Volpe's Neurology of the Newborn. , 275-321 (2018).</li><li>Shankaran, S., 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=Network+EKSNNR.+Childhood+outcomes+after+hypothermia+for+neonatal+encephalopathy.">Network EKSNNR. Childhood outcomes after hypothermia for neonatal encephalopathy.</a> New England Journal of Medicine. 366 (22), 2085-2092 (2012).</li><li>Pappas, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cognitive+outcomes+after+neonatal+encephalopathy.">Cognitive outcomes after neonatal encephalopathy.</a> Pediatrics. 135 (3), 624-634 (2015).</li><li>van Schie, P. E., 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=Long-term+motor+and+behavioral+outcome+after+perinatal+hypoxic-ischemic+encephalopathy.">Long-term motor and behavioral outcome after perinatal hypoxic-ischemic encephalopathy.</a> European Journal of Paediatric Neurology. 19 (3), 354-359 (2015).</li><li>Rensing, N., 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=Longitudinal+analysis+of+developmental+changes+in+electroencephalography+patterns+and+sleep-wake+states+of+the+neonatal+mouse.">Longitudinal analysis of developmental changes in electroencephalography patterns and sleep-wake states of the neonatal mouse.</a> PLoS One. 13 (11), 1-17 (2018).</li><li>Rice, J. E., Vannucci, R. C., Brierley, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+immaturity+on+hypoxic-ischemic+brain+damage+in+the+rat.">The influence of immaturity on hypoxic-ischemic brain damage in the rat.</a> Annals of Neurology. 9 (2), 131-141 (1981).</li><li>Burnsed, J. C., 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=Hypoxia-ischemia+and+therapeutic+hypothermia+in+the+neonatal+mouse+brain--a+longitudinal+study.">Hypoxia-ischemia and therapeutic hypothermia in the neonatal mouse brain--a longitudinal study.</a> PLoS One. 10 (3), 0118889 (2015).</li><li>Semple, B. D., 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=Brain+development+in+rodents+and+humans:+Identifying+benchmarks+of+maturation+and+vulnerability+to+injury+across+species.">Brain development in rodents and humans: Identifying benchmarks of maturation and vulnerability to injury across species.</a> Progress in Neurobiology. , 1-16 (2013).</li><li>Comi, A. M., 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=Gabapentin+neuroprotection+and+seizure+suppression+in+immature+mouse+brain+ischemia.">Gabapentin neuroprotection and seizure suppression in immature mouse brain ischemia.</a> Pediatric Research. 64 (1), 81-85 (2008).</li><li>Comi, A. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuous+Electroencephalographic+Monitoring+with+Radio-Telemetry+in+a+Rat+Model+of+Perinatal+Hypoxia-Ischemia+Reveals+Progressive+Post-Stroke+Epilepsy.">Continuous Electroencephalographic Monitoring with Radio-Telemetry in a Rat Model of Perinatal Hypoxia-Ischemia Reveals Progressive Post-Stroke Epilepsy.</a> Journal of Neuroscience. 30 (1), 404-415 (2010).</li><li>Burnsed, 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=Neuronal+Circuit+Activity+during+Neonatal+Hypoxic+-+Ischemic+Seizures+in+Mice.">Neuronal Circuit Activity during Neonatal Hypoxic - Ischemic Seizures in Mice.</a> Annals of Neurology. 86, 927-938 (2019).</li><li>Sampath, D., White, A. M., Raol, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+neonatal+seizures+in+an+animal+model+of+hypoxic-ischemic+encephalopathy.">Characterization of neonatal seizures in an animal model of hypoxic-ischemic encephalopathy.</a> Epilepsia. 55 (7), 985-993 (2014).</li><li>Sampath, D., Valdez, R., White, A. M., Raol, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anticonvulsant+effect+of+flupirtine+in+an+animal+model+of+neonatal+hypoxic-ischemic+encephalopathy.">Anticonvulsant effect of flupirtine in an animal model of neonatal hypoxic-ischemic encephalopathy.</a> Neuropharmacology. 123, 126-135 (2017).</li><li>Kang, S. K., 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=and+sex-dependent+susceptibility+to+phenobarbital-resistant+neonatal+seizures:+role+of+chloride+co-transporters.">and sex-dependent susceptibility to phenobarbital-resistant neonatal seizures: role of chloride co-transporters.</a> Frontiers in Cellular Neuroscience. 9, 1-16 (2015).</li><li>Zanelli, S., Goodkin, H. 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Hemos presentado un modelo para la monitorización continua de video-EEG en ratones neonatales durante las convulsiones hipóxico-isquémicas. El análisis de video en conjunto con EEG permite la caracterización de la semiología de convulsiones. El análisis de EEG permite la extracción de espectrogramas de potencia y el análisis de amplitud de fondo.
La colocación correcta y cuidadosa de los electrodos es crucial en este protocolo, ya que las lesiones durante la colocación de los electr...

La encefalopatía isquémica hipóxica (HIE) es una afección que afecta a 1,5 de cada 1000 recién nacidos anualmente y es la causa más frecuente de convulsiones neonatales1,2. Los bebés que sobreviven están en riesgo de diversas discapacidades neurológicas como parálisis cerebral, discapacidad intelectual y epilepsia3,4,5.
Los modelos animales juegan un papel crítico en la comprensión e investigación de la fisiopatología de la isquemia por hipoxia y las convulsiones neonatales6,7. Se utiliza un modelo de Vannucci modificado para inducir isquemia por hipoxia (HI) en el día 10 postnatal (p10)7,8. Los cachorros de ratón de esta edad se traducen neurológicamente aproximadamente al término completo neonato humano9.
La monitorización continua por video electroencefalografía (EEG) utilizada junto con este modelo de lesión permite una mayor comprensión y caracterización de las convulsiones isquémicas hipóxicas neonatales. Estudios previos han utilizado diversos métodos para analizar las convulsiones neonatales en roedores, incluidas grabaciones de video, grabaciones limitadas de EEG y grabaciones de EEG de telemetría10,11,12,13,14,15,16. En el siguiente manuscrito, discutimos en profundidad el proceso de grabación de video continuo EEG en cachorros de ratón durante la hipoxia-isquemia. Esta técnica para el monitoreo continuo de video EEG en cachorros de ratón neonatales podría aplicarse a una variedad de modelos de enfermedades y convulsiones.

<table>
	<tbody>
		<tr>
			<td>SURGERY</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ball Point Applicator</td>
			<td>Metrex Research</td>
			<td>8300-F</td>
			<td>i-bond applicator</td>
		</tr>
		<tr>
			<td>Cranioplast (Powder/Resin)</td>
			<td>Coltene</td>
			<td>H00383</td>
			<td>Perm Reline/Power</td>
		</tr>
		<tr>
			<td>I-Bond</td>
			<td>Kulzer GmbH, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LOOK Silk Suture</td>
			<td>Surgical Specialities Corporation</td>
			<td>SP115</td>
			<td>LOOK SP115 Black Braided Silk Non absorbable surgical suture</td>
		</tr>
		<tr>
			<td>RS-5168 Botvin Forceps</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS5168</td>
			<td>Forcep for surgery/ligation</td>
		</tr>
		<tr>
			<td>RS-5138 Graefe Forceps</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS5138</td>
			<td>Forcep for surgery/ligation</td>
		</tr>
		<tr>
			<td>UV light for I-Bond</td>
			<td>Blast Lite By First Media</td>
			<td>BL778</td>
			<td>UV ligth for I-bond</td>
		</tr>
		<tr>
			<td>Vannas Microdissecting Scissor</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS5618</td>
			<td>Scissor for ligation</td>
		</tr>
		<tr>
			<td>Vet Bond</td>
			<td>3M Vetbond</td>
			<td>1469SB</td>
			<td>Vet Glue</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HYPOXIA</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hypoxidial</td>
			<td>Starr Life Science</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Oxygen sensor</td>
			<td>Medical Products</td>
			<td></td>
			<td>MiniOxI- oxygen analyzer/sensor for hypoxia rig</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EEG RECORDING</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Female receptacle connector 0.079&#34;</td>
			<td>Mill-Max Manufacturing Corp</td>
			<td>832-10-024-10-001000</td>
			<td>Ordered from Digikey</td>
		</tr>
		<tr>
			<td>Grass Amplifier</td>
			<td>Natus Neurology Incorporated</td>
			<td></td>
			<td>Grass Product</td>
		</tr>
		<tr>
			<td>LabChart Pro</td>
			<td>ADI Instruments</td>
			<td></td>
			<td>Software to run the system</td>
		</tr>
		<tr>
			<td>Male Socket Connector 0.079&#34;</td>
			<td>Mill-Max Manufacturing Corp</td>
			<td>833-43-024-20-001000</td>
			<td>Ordered from Digikey</td>
		</tr>
		<tr>
			<td>Operational Amplifier</td>
			<td>Texas Instruments, Dallas, TX, USA</td>
			<td>TLC2274CD</td>
			<td>TLC2274 Quad Low&#8208;Noise Rail&#8208;to Rail Operational Amplifier</td>
		</tr>
		<tr>
			<td>Operational Amplifier</td>
			<td>Texas Instruments, Dallas, TX, USA</td>
			<td>TLC2272ACDR</td>
			<td>TLC2274 Quad Low&#8208;Noise Rail&#8208;to Rail Operational Amplifier</td>
		</tr>
		<tr>
			<td>Stainless Steel wire</td>
			<td>A-M Systems</td>
			<td>791400</td>
			<td>0.005&#34; Bare/0.008&#34; Coated 100 ft</td>
		</tr>
		<tr>
			<td>Ultra-Flexible Wire</td>
			<td>McMaster-Carr</td>
			<td>9564T1</td>
			<td>36 Gauze wire of various color</td>
		</tr>
	</tbody>
</table>

continuous video electroencephalogram during hypoxia-ischemia in neonatal mice

La isquemia por hipoxia es la causa más común de convulsiones neonatales. Los modelos animales son cruciales para comprender los mecanismos y la fisiología subyacentes a las convulsiones neonatales y la isquemia por hipoxia. Este manuscrito describe un método para el monitoreo continuo de video electroencefalograma (EEG) en ratones neonatales para detectar convulsiones y analizar el fondo de EEG durante la isquemia por hipoxia. El uso de video y EEG en conjunto permite la descripción de la semiología de convulsiones y la confirmación de convulsiones. Este método también permite el análisis de espectrogramas de potencia y tendencias de patrones de fondo de EEG durante el período de tiempo experimental. En este modelo de isquemia por hipoxia, el método permite el registro de EEG antes de la lesión para obtener una línea de base normativa y durante la lesión y la recuperación. El tiempo total de monitoreo está limitado por la incapacidad de separar a los cachorros de la madre durante más de cuatro horas. Aunque, hemos utilizado un modelo de convulsiones hipóxico-isquémicas en este manuscrito, este método para el monitoreo de EEG de video neonatal podría aplicarse a diversos modelos de enfermedades y convulsiones en roedores.

Semiología de convulsiones
La exposición a hipoxia-isquemia neonatal da lugar a convulsiones generalizadas y focales en ratones (Figura 1A-C). Las grabaciones de video EEG permiten que los hallazgos electrográficos se correlacionen con el comportamiento en video. Estos comportamientos se puntuaron utilizando una puntuación de convulsiones conductuales de roedores neonatales (BSS) publicada anteriormente16. Adem...

Watch this Scientific Journal Video about Continuous Video Electroencephalogram during Hypoxia-Ischemia in Neonatal Mice at JoVE.com

Continuous Video Electroencephalogram during Hypoxia-Ischemia in Neonatal Mice

Este manuscrito describe un método para grabaciones continuas de video EEG utilizando electrodos de profundidad múltiple en ratones neonatales sometidos a hipoxia-isquemia.

Electroencefalograma de video continuo durante la hipoxia-isquemia en ratones neonatales

Hypoxia ischemia is the most common cause of neonatal seizures. Animal models are crucial for understanding the mechanisms and physiology underlying ...

continuous-video-electroencephalogram-during-hypoxia-ischemia

Research

JoVE Journal

Neuroscience

3.3K vistas.  University of Virginia. Este manuscrito describe un método para grabaciones continuas de video EEG utilizando electrodos de profundidad múltiple en ratones neonatales sometidos a hipoxia-isquemia.

Video: Continuous Video Electroencephalogram during Hypoxia-Ischemia in Neonatal Mice

Video-oculography in Mice

Eye movements are very important in order to track an object or to stabilize an image on the retina during movement. Animals without a fovea, such as the mouse, have a limited capacity to lock their eyes onto a target. In contrast to these target directed eye movements, compensatory ocular eye movements are easily elicited in afoveate animals1,2,3,4. Compensatory ocular movements are generated by processing vestibular and optokinetic information into a command signal that will drive the eye muscles. The processing of the vestibular and optokinetic information can be investigated separately and together, allowing the specification of a deficit in the oculomotor system. The oculomotor system can be tested by evoking an optokinetic reflex (OKR), vestibulo-ocular reflex (VOR) or a visually-enhanced vestibulo-ocular reflex (VVOR). The OKR is a reflex movement that compensates for "full-field" image movements on the retina, whereas the VOR is a reflex eye movement that compensates head movements. The VVOR is a reflex eye movement that uses both vestibular as well as optokinetic information to make the appropriate compensation. The cerebellum monitors and is able to adjust these compensatory eye movements. Therefore, oculography is a very powerful tool to investigate brain-behavior relationship under normal as well as under pathological conditions (f.e. of vestibular, ocular and/or cerebellar origin).
Testing the oculomotor system, as a behavioral paradigm, is interesting for several reasons. First, the oculomotor system is a well understood neural system5. Second, the oculomotor system is relative simple6; the amount of possible eye movement is limited by its ball-in-socket architecture ("single joint") and the three pairs of extra-ocular muscles7. Third, the behavioral output and sensory input can easily be measured, which makes this a highly accessible system for quantitative analysis8. Many behavioral tests lack this high level of quantitative power. And finally, both performance as well as plasticity of the oculomotor system can be tested, allowing research on learning and memory processes9.
Genetically modified mice are nowadays widely available and they form an important source for the exploration of brain functions at various levels10. In addition, they can be used as models to mimic human diseases. Applying oculography on normal, pharmacologically-treated or genetically modified mice is a powerful research tool to explore the underlying physiology of motor behaviors under normal and pathological conditions. Here, we describe how to measure video-oculography in mice8.

Video-oculography is a very quantitative method to investigate ocular motor performance as well as motor learning. Here, we describe how to measure video-oculography in mice. Applying this technique on normal, pharmacologically-treated or genetically modified mice is a powerful research tool to explore the underlying physiology of motor behaviors.

Vídeo-oculografía en ratones

Eye movements are very important in order to track an object or to stabilize an image on the retina during movement. Animals without a fovea, such as the ...

Investigación

Neurociencias

Neonatal Subventricular Zone Electroporation

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal cells1. Understanding the molecular pathways responsible for NSC self-renewal, commitment, and differentiation is critical for harnessing their unique potential to repair the brain and better understand central nervous system disorders. Previous methods for the manipulation of mammalian systems required the time consuming and expensive endeavor of genetic engineering at the whole animal level2. Thus, the vast majority of studies have explored the functions of NSC molecules in vitro or in invertebrates.
Here, we demonstrate the simple and rapid technique to manipulate neonatal NPCs that is referred to as neonatal subventricular zone (SVZ) electroporation. Similar techniques were developed a decade ago to study embryonic NSCs and have aided studies on cortical development3,4 . More recently this was applied to study the postnatal rodent forebrain5-7. This technique results in robust labeling of SVZ NSCs and their progeny. Thus, postnatal SVZ electroporation provides a cost and time effective alternative for mammalian NSC genetic engineering.

We demonstrate a minimally invasive technique referred to as neonatal subventricular zone electroporation. The technique consists of injecting plasmid DNA into the lateral ventricles of neonatal pups and applying electrical current to deliver and genetically manipulate neural stem cells

La electroporación neonatal zona subventricular

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal ...

Neonatal Pial Surface Electroporation

Over the past several years the pial surface has been identified as a germinal niche of importance during embryonic, perinatal and adult neuro- and gliogenesis, including after injury. However, methods for genetically interrogating these progenitor populations and tracking their lineages had been limited owing to a lack of specificity or time consuming production of viruses. Thus, progress in this region has been relatively slow with only a handful of investigations of this location. Electroporation has been used for over a decade to study neural stem cell properties in the embryo, and more recently in the postnatal brain. Here we describe an efficient, rapid, and simple technique for the genetic manipulation of pial surface progenitors based on an adapted electroporation approach. Pial surface electroporation allows for facile genetic labeling and manipulation of these progenitors, thus representing a time-saving and economical approach for studying these cells.

The pial surface is a unique progenitor zone in the CNS that is receiving increasing attention. Herein, we detail a method for rapid genetic manipulation of this progenitor zone using a modified electroporation method. This procedure can be used for cellular and molecular investigations of cell lineages and signaling pathways involved in cell differentiation and to elucidate the fate and properties of daughter cells.

Neonatal electroporación superficie pial

Over the past several years the pial surface has been identified as a germinal niche of importance during embryonic, perinatal and adult neuro- and ...

Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene expression in the mouse brain. Here we describe a technique first introduced by Passini and Wolfe for direct intracranial delivery of virally-encoded transgenes into the neonatal mouse brain. In its most basic form, the procedure requires only an ice bucket and a microliter syringe. However, the protocol can also be adapted for use with stereotaxic frames to improve consistency for researchers new to the technique. The method relies on the ability of adeno-associated virus (AAV) to move freely from the cerebral ventricles into the brain parenchyma while the ependymal lining is still immature during the first 12-24 hr after birth. Intraventricular injection of AAV at this age results in widespread transduction of neurons throughout the brain. Expression begins within days of injection and persists for the lifetime of the animal. Viral titer can be adjusted to control the density of transduced neurons, while co-expression of a fluorescent protein provides a vital label of transduced cells. With the rising availability of viral core facilities to provide both off-the-shelf, pre-packaged reagents and custom viral preparation, this approach offers a timely method for manipulating gene expression in the mouse brain that is fast, easy, and far less expensive than traditional germline engineering.

Here we demonstrate a technique for widespread neuronal transduction by intraventricular injection of adeno-associated virus into the neonatal mouse brain. This method provides a rapid and easy way to attain lifelong expression of virally-delivered transgenes.

Inyección Viral Intracerebroventricular de Neonatología del ratón Cerebro de transducción neuronal persistente y generalizada

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

Un método simple y barato para la Determinación de la sensibilidad al frío y Adaptación en ratones

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein isa multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

Utilizando metodologías combinadas para definir el papel de la membrana plasmática de entrega Durante Axon ramas y morfogénesis neuronal

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

Video Imaging and Spatiotemporal Maps to Analyze Gastrointestinal Motility in Mice

The enteric nervous system (ENS) plays an important role in regulating gastrointestinal (GI) motility and can function independently of the central nervous system. Changes in ENS function are a major cause of GI symptoms and disease and may contribute to GI symptoms reported in neuropsychiatric disorders including autism. It is well established that isolated colon segments generate spontaneous, rhythmic contractions known as Colonic Migrating Motor Complexes (CMMCs). A procedure to analyze the enteric neural regulation of CMMCs in ex vivo preparations of mouse colon is described. The colon is dissected from the animal and flushed to remove fecal content prior to being cannulated in an organ bath. Data is acquired via a video camera positioned above the organ bath and converted to high-resolution spatiotemporal maps via an in-house software package. Using this technique, baseline contractile patterns and pharmacological effects on ENS function in colon segments can be compared over 3-4 hr. In addition, propagation length and speed of CMMCs can be recorded as well as changes in gut diameter and contraction frequency. This technique is useful for characterizing gastrointestinal motility patterns in transgenic mouse models (and in other species including rat and guinea pig). In this way, pharmacologically induced changes in CMMCs are recorded in wild type mice and in the Neuroligin-3R451C mouse model of autism. Furthermore, this technique can be applied to other regions of the GI tract including the duodenum, jejunum and ileum and at different developmental ages in mice.

This article describes a video imaging technique and high-resolution spatiotemporal mapping to identify changes in the neural regulation of colonic motility in adult mice. Subtle effects on gastrointestinal (GI) function can be detected using this approach in isolated tissue preparations to advance our understanding of GI disease.

Imágenes de vídeo y mapas espacio-temporales para Analizar la motilidad gastrointestinal en ratones

The enteric nervous system (ENS) plays an important role in regulating gastrointestinal (GI) motility and can function independently of the central ...

Recording EEG in Freely Moving Neonatal Rats Using a Novel Method

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such as epilepsy and neurodegenerative disorders. However, it is technically difficult to obtain EEG recordings in neonates as it requires specialized handling and great care. Here, we present a novel method to record EEG in neonatal rat pups (P8-P15). We designed a simple and reliable electrode using computer pin loci; it can be easily implanted into the skull of a rat pup to record high-quality EEG signals in the normal and epileptic brain. Pups were given an intraperitoneal (i.p.) injection of the neurotoxin kainic acid (KA) to induce epileptic seizures. The surgical implantation performed in this procedure is less expensive than other EEG procedures for neonates. This method allows one to record high-quality and stable EEG signals for more than 1 week. Furthermore, this procedure can also be applied to adult rats and mice to study epilepsy or other neurological disorders.

Here, we introduce a novel technique designed to record electroencephalography (EEG) in freely moving neonatal epileptic pups and describe its procedures, features, and applications. This method allows one to record EEG for more than 1 week.

Grabación de EEG en Ratas Neonatales que se mueven libremente usando un método nuevo

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such ...

Spinal Cord Neurons Isolation and Culture from Neonatal Mice

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on postnatal day 1-3. A mouse litter, usually 4-10 pups born from one breeding pair, is gathered for one experiment, and spinal cords are collected individually from each mouse after euthanasia with isoflurane. The spinal column is dissected out and then the spinal cord is released from the column. The spinal cords are then minced to increase the surface area of delivery for an enzymatic protease that allows for the neurons and other cells to be released from the tissue. Trituration is then used to release the cells into solution. This solution is subsequently fractionated in a density gradient to separate the various cells in solution, allowing for neurons to be isolated. Approximately 1-2.5 x 106 neurons can be isolated from one litter group. The neurons are then seeded onto wells coated with adhesive factors that allow for proper growth and maturation. The neurons take approximately 7 days to reach maturity in the growth and culture medium and can be used thereafter for treatment and analysis.

This study presents a technique for the isolation of neurons from WT neonatal mice. It requires the careful dissection of the spinal cord from the neonatal mouse, followed by the separation of neurons from the spinal cord tissue through mechanical and enzymatic cleavage.

Aislamiento y Cultura de Neuronas de la Médula Espinal de Ratones Neonatales

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

En Vivo Proyección de imagen de dos fotones de neuronas corticales en ratones neonatales

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...