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. 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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.

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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

Universidad de Cartagena UDEC

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

Delivery of In Vivo Acute Intermittent Hypoxia in Neonatal Rodents to Prime Subventricular Zone-derived Neural Progenitor Cell Cultures

Extended culture of neural stem/progenitor cells facilitates in vitro analyses to understand their biology while enabling expansion of cell populations to adequate numbers prior to transplantation. Identifying approaches to refine this process, to augment the production of all CNS cell types (i.e., neurons), and to possibly contribute to therapeutic cell therapy protocols is a high research priority. This report describes an easily applied in vivo &#8220;pre-conditioning&#8221; stimulus which can be delivered to awake, non-anesthetized animals. Thus, it is a non-invasive and non-stressful procedure. Specifically described are the procedures for exposing mouse or rat pups (aged postnatal day 1-8) to a brief (40-80 min) period of intermittent hypoxia (AIH). The procedures included in this video protocol include calibration of the whole-body plethysmography chamber in which pups are placed during AIH and the technical details of AIH exposure. The efficacy of this approach to elicit tissue-level changes in the awake animal is demonstrated through the enhancement of subsequent in vitro expansion and neuronal differentiation in cells harvested from the subventricular zone (SVZ). These results support the notion that tissue level changes across multiple systems could be observed following AIH, and support the continued optimization and establishment of AIH as a priming or conditioning modality for therapeutic cell populations.

Delivery of In Vivo Acute Intermittent Hypoxia in Neonatal Rodents to Prime Subventricular Zone-derived Neural Progenitor Cell Cultures

This article describes the methodology for administering short periods of intermittent hypoxia to postnatal day 1-8 mouse or rat pups. This approach effectively elicits a robust tissue level &#8220;priming effect&#8221; on cultured neural progenitor cells that are harvested within 30 min of hypoxia exposure.

Entrega de<em&gt; En Vivo</em&gt; Aguda intermitente hipoxia neonatal en roedores a derivados de la Zona Prime subventricular progenitoras neurales Cultivos Celulares

Extended culture of neural stem/progenitor cells facilitates in vitro analyses to understand their biology while enabling expansion of cell populations to ...

Investigación

Biología del Desarrollo

A Swine Model of Neonatal Asphyxia

Annually more than 1 million neonates die worldwide as related to asphyxia. Asphyxiated neonates commonly have multi-organ failure including hypotension, perfusion deficit, hypoxic-ischemic encephalopathy, pulmonary hypertension, vasculopathic enterocolitis, renal failure and thrombo-embolic complications. Animal models are developed to help us understand the patho-physiology and pharmacology of neonatal asphyxia. In comparison to rodents and newborn lambs, the newborn piglet has been proven to be a valuable model. The newborn piglet has several advantages including similar development as that of 36-38 weeks human fetus with comparable body systems, large body size (&tilde;1.5-2 kg at birth) that allows the instrumentation and monitoring of the animal and controls the confounding variables of hypoxia and hemodynamic derangements.
We here describe an experimental protocol to simulate neonatal asphyxia and allow us to examine the systemic and regional hemodynamic changes during the asphyxiating and reoxygenation process as well as the respective effects of interventions. Further, the model has the advantage of studying multi-organ failure or dysfunction simultaneously and the interaction with various body systems. The experimental model is a non-survival procedure that involves the surgical instrumentation of newborn piglets (1-3 day-old and 1.5-2.5 kg weight, mixed breed) to allow the establishment of mechanical ventilation, vascular (arterial and central venous) access and the placement of catheters and flow probes (Transonic Inc.) for the continuously monitoring of intra-vascular pressure and blood flow across different arteries including main pulmonary, common carotid, superior mesenteric and left renal arteries. Using these surgically instrumented piglets, after stabilization for 30-60 minutes as defined by Z&lt;10% variation in hemodynamic parameters and normal blood gases, we commence an experimental protocol of severe hypoxemia which is induced via normocapnic alveolar hypoxia. The piglet is ventilated with 10-15% oxygen by increasing the inhaled concentration of nitrogen gas for 2h, aiming for arterial oxygen saturations of 30-40%. This degree of hypoxemia will produce clinical asphyxia with severe metabolic acidosis, systemic hypotension and cardiogenic shock with hypoperfusion to vital organs. The hypoxia is followed by reoxygenation with 100% oxygen for 0.5h and then 21% oxygen for 3.5h. Pharmacologic interventions can be introduced in due course and their effects investigated in a blinded, block-randomized fashion.

Large animal models have good translational values in the examination of physiology and pharmacology of neonatal asphyxia. Using newborn piglets, we develop an experimental protocol to simulate neonatal asphyxia which has advantages of studying the systemic and regional hemodynamics, oxygen transport with biochemical and pathologic pathways and correlations.

Un modelo porcino de la asfixia neonatal

Annually more than 1 million neonates die worldwide as related to asphyxia. Asphyxiated neonates commonly have multi-organ failure including hypotension, ...

Medicina

Simultaneous Video-EEG-ECG Monitoring to Identify Neurocardiac Dysfunction in Mouse Models of Epilepsy

In epilepsy, seizures can evoke cardiac rhythm disturbances such as heart rate changes, conduction blocks, asystoles, and arrhythmias, which can potentially increase risk of sudden unexpected death in epilepsy (SUDEP). Electroencephalography (EEG) and electrocardiography (ECG) are widely used clinical diagnostic tools to monitor for abnormal brain and cardiac rhythms in patients. Here, a technique to simultaneously record video, EEG, and ECG in mice to measure behavior, brain, and cardiac activities, respectively, is described. The technique described herein utilizes a tethered (i.e., wired) recording configuration in which the implanted electrode on the head of the mouse is hard-wired to the recording equipment. Compared to wireless telemetry recording systems, the tethered arrangement possesses several technical advantages such as a greater possible number of channels for recording EEG or other biopotentials; lower electrode costs; and greater frequency bandwidth (i.e., sampling rate) of recordings. The basics of this technique can also be easily modified to accommodate recording other biosignals, such as electromyography (EMG) or plethysmography for assessment of muscle and respiratory activity, respectively. In addition to describing how to perform the EEG-ECG recordings, we also detail methods to quantify the resulting data for seizures, EEG spectral power, cardiac function, and heart rate variability, which we demonstrate in an example experiment using a mouse with epilepsy due to Kcna1 gene deletion. Video-EEG-ECG monitoring in mouse models of epilepsy or other neurological disease provides a powerful tool to identify dysfunction at the level of the brain, heart, or brain-heart interactions.

Here, we present a protocol to record brain and heart bio signals in mice using simultaneous video, electroencephalography (EEG), and electrocardiography (ECG). We also describe methods to analyze the resulting EEG-ECG recordings for seizures, EEG spectral power, cardiac function, and heart rate variability.

Video-EEG-ECG simultánea de monitoreo para identificar la disfunción neurocardiaco en modelos de ratón de la epilepsia

In epilepsy, seizures can evoke cardiac rhythm disturbances such as heart rate changes, conduction blocks, asystoles, and arrhythmias, which can ...

Genética

The Hypoxic Ischemic Encephalopathy Model of Perinatal Ischemia

Hypoxic-Ischemic Encephalopathy (HIE) is the consequence of systemic asphyxia occurring at birth. Twenty five percent of neonates with HIE develop severe and permanent neuropsychological sequelae, including mental retardation, cerebral palsy, and epilepsy. The outcomes of HIE are devastating and permanent, making it critical to identify and develop therapeutic strategies to reduce brain injury in newborns with HIE. To that end, the neonatal rat model for hypoxic-ischemic brain injury has been developed to model this human condition. The HIE model was first validated by Vannucci et al 1 and has since been extensively used to identify mechanisms of brain injury resulting from perinatal hypoxia-ischemia 2 and to test potential therapeutic interventions 3,4. The HIE model is a two step process and involves the ligation of the left common carotid artery followed by exposure to a hypoxic environment. Cerebral blood flow (CBF) in the hemisphere ipsilateral to the ligated carotid artery does not decrease because of the collateral blood flow via the circle of Willis; however with lower oxygen tension, the CBF in the ipsilateral hemisphere decreases significantly and results in unilateral ischemic injury. The use of 2,3,5-triphenyltetrazolium chloride (TTC) to stain and identify ischemic brain tissue was originally developed for adult models of rodent cerebral ischemia 5, and is used to evaluate the extent of cerebral infarctin at early time points up to 72 hours after the ischemic event 6. In this video, we demonstrate the hypoxic-ischemic injury model in postnatal rat brain and the evaluation of the infarct size using TTC staining.

The postnatal rat model for hypoxic-ischemic brain injury is a well-established model of human neonatal hypoxic ischemic encephalopathy (HIE). In this article, we describe the model of HIE in post-natal rat pups.

El modelo de encefalopatía isquémica hipóxica de la isquemia perinatal

Hypoxic-Ischemic Encephalopathy (HIE) is the consequence of systemic asphyxia occurring at birth. Twenty five percent of neonates with HIE develop severe ...

Biología

A Piglet Model of Neonatal Hypoxic-Ischemic Encephalopathy

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

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

Un modelo del cochinillo de Neonatología encefalopatía hipóxico-isquémica

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

Neurobehavioral Assessments in a Mouse Model of Neonatal Hypoxic-ischemic Brain Injury

We performed unilateral carotid artery occlusion on CD-1 mice to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of neonatal HI brain injury by studying neurobehavioral functions in these mice compared to non-operated (i.e., normal) mice. During the study, Rice-Vannucci&#39;s method was used to induce neonatal HI brain damage in postnatal day 7-10 (P7-10) mice. The HI operation was performed on the pups by unilateral carotid artery ligation and exposure to hypoxia (8% O2 and 92% N2 for 90 min). One week after the operation, the damaged brains were evaluated with the naked eye through the semi-transparent skull and were categorized into subgroups based on the absence (&#34;no cortical injury&#34; group) or presence (&#34;cortical injury&#34; group) of cortical injury, such as a lesion in the right hemisphere. On week 6, the following neurobehavioral tests were performed to evaluate the cognitive and motor functions: passive avoidance task (PAT), ladder walking test, and grip strength test. These behavioral tests are helpful in determining the effects of neonatal HI brain injury and are used in other mouse models of neurodegenerative diseases. In this study, neonatal HI brain injury mice showed motor deficits that corresponded to right hemisphere damage. The behavioral test results are relevant to the deficits observed in human neonatal HI patients, such as cerebral palsy or neonatal stroke patients. In this study, a mouse model of neonatal HI brain injury was established and showed different degrees of motor deficits and cognitive impairment compared to non-operated mice. This work provides basic information on the HI mouse model. MRI images demonstrate the different phenotypes, separated according to the severity of brain damage by motor and cognitive tests.

We performed unilateral carotid artery occlusion on postnatal day 7-10 CD-1 mouse pups to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of HI brain injury. We studied neurobehavioral functions in these mice compared to non-operated normal mice.

Evaluación neuroconductual en un modelo murino de lesión de cerebro hipóxico-isquémica Neonatal

We performed unilateral carotid artery occlusion on CD-1 mice to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of neonatal HI ...

Neurociencias

Noninvasive Electrocardiography in the Perinatal Mouse

Electrocardiography (ECG) has long been relied upon as an effective and reliable method of assessing cardiovascular (and cardiopulmonary) function in both human and animal models of disease. Individual heart rate, rhythm, and regularity, combined with quantitative parameters collected from ECG, serve to assess the integrity of the cardiac conduction system as well as the integrated physiology of the cardiac cycle. This article provides a comprehensive description of the methods and techniques used to perform a noninvasive ECG on perinatal and neonatal mouse pups as early as the first postnatal day, without requiring the use of anesthetics. This protocol was designed to directly address a need for a standardized and repeatable method for obtaining ECG in newborn mice. From a translational perspective, this protocol proves to be entirely effective for characterization of congenital cardiopulmonary defects generated using transgenic mouse lines, and particularly for analysis of defects causing lethality at or during the first postnatal days. This protocol also aims to directly address a gap in the scientific literature to characterize and provide normative data associated with maturation of the early postnatal cardiac conduction system. This method is not limited to a specific postnatal timepoint, but rather allows for ECG data collection in neonatal mouse pups from birth to postnatal day 10 (P10), a window that is of critical importance for modeling human diseases in vivo, with particular emphasis on congenital heart disease (CHD).

Here, we present a noninvasive electrocardiography (ECG) protocol, optimized for early postnatal mice, that does not require the use of anesthetics.

Electrocardiografía no invasiva en el ratón perinatal

Electrocardiography (ECG) has long been relied upon as an effective and reliable method of assessing cardiovascular (and cardiopulmonary) function in both ...

Echocardiographic Characterization of Left Ventricular Structure, Function, and Coronary Flow in Neonate Mice

Echocardiography is a non-invasive procedure that enables the evaluation of structural and functional parameters in animal models of cardiovascular disease and is used to assess the impact of potential treatments in preclinical studies. Echocardiographic studies are usually conducted in young adult mice (i.e., 4-6 weeks of age). The evaluation of early neonatal cardiovascular function is not usually performed because of the small size of the mouse pups and the associated technical difficulties. One of the most important challenges is that the short length of the pups&#39; limbs prevents them from reaching the electrodes in the echocardiography platform. Body temperature is the other challenge, as pups are very susceptible to changes in temperature. Therefore, it is important to establish a practical guide for performing echocardiographic studies in small mouse pups to help researchers detect early pathological changes and study the progression of cardiovascular disease over time. The current work describes a protocol for performing echocardiography in mouse pups at the early age of 7 days old. The echocardiographic characterization of cardiac morphology, function, and coronary flow in neonatal mice is also described.

The present protocol describes the echocardiographic assessment of left ventricular morphology, function, and coronary blood flow in 7-day old neonate mice.

Caracterización ecocardiográfica de la estructura, función y flujo coronario del ventrículo izquierdo en ratones neonatos

Echocardiography is a non-invasive procedure that enables the evaluation of structural and functional parameters in animal models of cardiovascular ...

Early Pathological and Magnetic Resonance Detection of Cerebral Injury Using a Rat Model of Neonatal Hypoxic Ischemic Encephalopathy

Perinatal hypoxic-ischemic encephalopathy (HIE) is an acute disease that may afflict newborns, resulting in variable long- and short-term neurodevelopmental outcomes. Early diagnosis is critical to identifying infants who may benefit from intervention; however, early diagnosis relies heavily on clinical criteria. No molecular or radiological tests have shown promise in detecting early cerebral injury. Studies have shown that magnetic resonance imaging (MRI) can show changes in both blood flow/ischemia and metabolic disruption. However, they have all been used to evaluate the secondary phase of the disease (&gt;12 h) after the onset of the injury. Early diagnosis is critical to rapidly starting therapeutic hypothermia in eligible infants, which is currently recommended to be initiated within 6 h of birth. The rat model of hypoxic-ischemic injury was developed in 1981 and has been validated and used extensively to study changes in brain perfusion, cerebral injury markers, and morphology. However, it has primarily been used as a "late model", evaluating injury several days after the initial ischemic insult. The model has been known to have poor sensitivity in evaluating reliable and reproducible early cerebral changes. The objective of this study was to develop a reliable model to study early gross morphological and radiological markers of HIE using pathological staining and cerebral magnetic resonance imaging/magnetic resonance spectroscopy.

The present protocol describes a rodent model of newborn hypoxic-ischemic injury for identifying early changes in cerebral tissue by gross morphology and magnetic resonance imaging. This has benefits over existing models, which can be used to study late injury but do not allow the evaluation of reproducible early changes.

Perinatal hypoxic-ischemic encephalopathy (HIE) is an acute disease that may afflict newborns, resulting in variable long- and short-term ...

Establishing a Mouse Model of Neonatal Hypoxic-Ischemic Brain Injury

Source: Kim, M., et al. <a href="https://appjove.unicartagenaproxy.elogim.com/v/55838">Neurobehavioral Assessments in a Mouse Model of Neonatal Hypoxic-ischemic Brain Injury.</a> J. Vis. Exp. (2017).
The video demonstrates the procedure for creating a mouse model to study neonatal hypoxic-ischemic brain injury. It outlines the steps for surgically inducing ischemia and subjecting the animal to hypoxia to exacerbate brain damage. Finally, it shows how to assess the resulting brain lesions, ensuring the mouse model is prepared for further scientific investigation.

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

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