Name: delivery of in vivo acute intermittent hypoxia in neonatal rodents to prime subventricular zone-derived neural progenitor cell cultures
Uploaded: 2015-11-02T15:00:00.000+00:00
Duration: 5 min 45 s
Description: Watch this Scientific Journal Video about Delivery of In Vivo Acute Intermittent Hypoxia in Neonatal Rodents to Prime Subventricular Zone-derived Neural Progenitor Cell Cultures at JoVE.com

The authors acknowledge funding sources responsible for this work: 5K12HD055929 (HHR), 5R01NS080180-02 (DDF).

NOTA: Todos los animales procedimientos en este protocolo se llevan a cabo con la aprobación de la Universidad de Florida Institucional Cuidado de Animales y el empleo Comisión (IACUC) y están en cumplimiento de la &#39;Guía para el Cuidado y Uso de Animales de Laboratorio &quot;. 1. Experimental básico establecido antes de la administración intermitente Hipoxia <ol><li> Expose cachorros 12 a las diferentes mezclas de gas usando un conjunto de cuerpo cámaras de pletismógrafo ratón en mucho la misma manera que los roedores adultos para otros fines experimentales 5,13,14. Las cámaras tienen un diámetro de 4 pulgadas (volumen = 450 ml), que es de tamaño amplio para acomodar 3-4 crías de ratón o 1-2 crías de rata. Aquí, los protocolos de HAI se entregan a los recién nacidos el día postnatal 1-8 (P1-P8) años. </li><li> Utilice los tanques de gas de aire comprimido para entregar el gas &quot;línea de base&quot; 21%, lo que mantiene la oxigenación de cámara en un 21%. Para lograr la &quot;hipoxia relativa&quot;, utilice un tanque de gas nitrógeno 10% de oxígeno / 90%. NOE: Los tubos de plástico (3/16 &quot;de diámetro) conecta los tanques de gas para un medidor de flujo. Por lo tanto, la entrada al medidor de flujo consiste en un tubo para cada línea de base y el flujo de aire hipoxia. </li><li> Configure el tubo de gas de la siguiente manera. <ol><li> Ejecutar tubo de plástico (1/8 &quot;de diámetro) desde el medidor de flujo a un regulador de flujo de sesgo, que permite a los flujos de 1 L / min a cada cámara (es decir, 4 L / min si se utilizan cámaras 4). </li><li> Ejecutar un tubo de plástico (1/8 &quot;de diámetro) del regulador de flujo sesgo a las cámaras de pletismografía. El flujo de aire a 1 L / min a cada cámara es una velocidad de flujo predeterminada a la que se considera el intercambio de gases a ser no de inducción de estrés a los animales sobre la base de observaciones fisiológicas y de comportamiento. </li></ol></li><li> Selle todas las conexiones no utilizadas hacia y desde la unidad de flujo sesgo y todas las aberturas no utilizadas a las cámaras con tapas de manera que no hay flujo de gas se escapa a otras cámaras no están en uso, o fuera de las cámaras de pletismografía durante el protocolo. </li><li> Coloque la cámara (s) En una incubadora antes de poner los animales en la cámara para permitir el equilibrio a 37 ° C, o temperatura corporal. </li></ol> 2. Calibración de los tiempos de ciclo de hipoxia intermitente <ol><li> Inspeccione todos los tubos, entre los tanques de aire y medidor de flujo, el flujo unidad de metro y la unidad de flujo de sesgo, y el flujo de sesgo y la cámara (s) pletismografía para las conexiones adecuadas. Las conexiones se configuran como se describe en el Paso 1. Verifique la conexión de un control gradual de todas las líneas y los cierres descritos: por ejemplo, la comprobación de líneas no utilizadas lejos de la unidad de flujo de sesgo y las aberturas de la cámara no esté en uso. </li><li> Conecte un sensor de ambiente O2 con un O 2 metros de mano y luego conecte el sensor a la tapa de la cámara pletismografía. </li><li> Abra las dos tanques de gas y conecte un par de pinzas quirúrgicas, de mango largo aislados con tubo de plástico para sujetar el flujo fuera de ciclo de gas. De esta manera, sólo un tanque de gas a la vez es la entrega de la 1 L / min de flujo de gas por cámara. Sujete la mezcla de gas &quot;línea de base&quot;, mientras que la mezcla de gas &quot;hipóxica&quot; se entrega a la cámara, y viceversa. </li><li> Registre el tiempo requerido para la cámara de O 2 para llegar a los niveles objetivo de 10% y volver a 21%. Utilizar los tiempos registrados para la entrega de protocolo subsiguiente. </li></ol> 3. Administración de aguda intermitente Hipoxia <ol><li> Inspeccionar todos los tubos y las conexiones como se describe en el Paso 2.1. </li><li> Coloca los recién nacidos en la cámara (s) pletismografía y albergar a las tapas de la cámara pletismografía en la base de la cámara. Confirmar un sello propio de la junta tórica es así como el cierre de las conexiones de cámara no utilizadas. Estos pasos son fundamentales para garantizar la prestación adecuada de mezclas de gases. </li><li> Coloque las cámaras de la celebración de los cachorros para ser tratados en la incubadora a 37 ° y cierran la puerta. Permitir que la cámara se equilibre a 37 ° C antes de iniciar el protocolo. Mantener los animales de control a 37 ° C en el incubato en la sala de la oxigenación del aire. </li><li> Abra las dos tanques de gas y el uso de un par de pinzas quirúrgicas, de mango largo aislados con tubo de plástico para sujetar el flujo fuera de ciclo de gas. De esta manera, sólo un tanque de gas a la vez es la entrega de la 1 L / min de flujo de gas por cámara. </li><li> Use un cronómetro de mano para, precisamente, ciclos de tiempo y para indicar el momento de cambiar las pinzas hemostáticas entre tubos, lo que resulta en la alternancia de flujo de gas a las cámaras. </li><li> Registre la terminación de cada ciclo usando un registro de tratamiento tal como el proporcionado en la Figura 1 para marcar todos los pasos del 2-paso, protocolo de ciclo de 20. </li><li> Monitorear la temperatura de la incubadora en cada cambio de ciclo para asegurar que los animales se mantienen en el rango señalado (33-35 ° C, que es el ajuste en la incubadora que se traduce en 37 ° C). </li><li> Seguir de cerca la actividad de las crías durante todo el protocolo para asegurar que los animales toleran ciclos sin angustia visible como vocalizaciones, alteración del nivel deactividad de la integridad física o la rodadura. </li></ol> 4. Aislamiento y cultivo de células madre / progenitoras de células de la zona subventricular NOTA: El cambio en la formación neuroesfera siguiente AIH comparación con los controles es un ejemplo de un punto final que demuestra la eficacia de este protocolo para provocar cambios en tejidos y nivel celular. <ol><li> Para aislar neuroesferas forman células, retire ratón o rata crías de la cámara inmediatamente después de HAI se ha completado. Sacrificio de los animales de acuerdo a los protocolos del IACUC dentro de los 30 min de terminación de protocolo. </li><li> Recoger el tejido SVZ, coloque en la cultura neuroesfera y llevar a cabo el paso y la expansión de neuroesferas derivados estándar, según lo publicado previamente en métodos capítulos 15,16 y un protocolo de video por separado 8. </li></ol>

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The authors have nothing to disclose.

This work reports the development of a protocol to expose neonatal rodents to AIH. The parameters described here effectively alter in situ neural stem cell biology, which is observable over several rounds of cell passage. Specifically, AIH increases the number of non-adherent neurospheres, the expansion of cells within each neurosphere (refected by sphere diameter), the expansion of adherent NPC populations, and the presence of neuroblasts in both non-adherent and adherent populations. It should be emph...

El objetivo de este método es para entregar de forma reproducible y eficaz episodios intermitentes de oxígeno ambiental sistémica rebajado a los roedores neonatales. La justificación del uso de la hipoxia intermitente (IH) para manipular la biología de células madre se origina a partir de experimentos de cultivo celular in vitro en el que se altera O 2 contenido del medio de crecimiento. En concreto, si se compara con las condiciones &quot;estándar&quot; de 20% de O 2, cultivo prolongado de células madre / progenitoras células poblaciones celulares en 3% O 2 resultados en un aumento de la proliferación, la disminución de la apoptosis y el aumento de 1,2 rendimiento neuronal. Este grupo tiene una gran experiencia con la administración de IH sistémica, y ha realizado extensos estudios sobre el papel de IH en la plasticidad respiratoria 3-7. Este trabajo, y el hallazgo reciente que IH crónica aumenta la neurogénesis en el roedor CNS 8-10, constituye la base para la exploración de aguda in vivohipoxia como estímulo acondicionamiento previo (es decir., antes de la cosecha de tejidos) en el posterior cultivo de células madre / progenitoras neuronales (CPN) 11. Sorprendentemente, cuando crías de ratón fueron expuestos a un breve período (&lt;1 hr) de la hipoxia intermitente aguda (AIH), las células que fueron cosechadas de la zona subventricular (SVZ) habían aumentado significativamente la capacidad para la expansión como neuroesferas o monocapa de células adherentes. El protocolo AIH también se asoció con un aumento de la expresión de un factor de &quot;destino neuronal&quot; transcripción (Pax6). En consecuencia, en vivo protocolos AIH pueden proporcionar un medio para &quot;prime&quot; NPCs antes del cultivo. Por ejemplo, las solicitudes de este enfoque podrían incluir la expansión de poblaciones de células antes del trasplante en el sistema nervioso central lesionado, o el simple aumento de la diferenciación neuronal de células cultivadas antes de los experimentos in vitro. Además, dado que se trata de una administración sistémica, cualquierórgano, tejido o célula es un candidato para el estudio similar. Por lo tanto, el protocolo como escrito es potencialmente aplicable a una amplia gama de estudios sobre la manipulación de oxígeno intermitente en pequeños mamíferos. Hay ciertas ventajas de este enfoque. En otro trabajo publicado, los neonatos fueron tratados como una camada con la presa en cámaras hipobárica, que permite la dosificación crónica, menos manipulación antes del tratamiento, y se mantienen contactos materna durante el tratamiento 9. El enfoque actual no pasa por tratamientos repetidos a una hembra reproductora, o el uso de una presa diferente para cada experimento. Este protocolo también permite el estudio de los recién nacidos de basura de concordancia y de la misma edad precisas. Los datos representativos demuestran otra fortaleza clave de este protocolo, es decir, la rapidez con la que AIH, como entregado, provoca una respuesta biológica potente y consistente en la biología de las células madre neurales. Esto establece un precedente para este protocolo para obtener biologi tejidos y nivel celularCal cambios que alteran la biología celular. Este informe se esbozarán los procedimientos detallados utilizados para la exposición de las crías de roedores a AIH, así como el análisis de la población de las células SVZ crecido como neuroesferas.

<table><tbody><tr><td>Mouse plethysmography chambers</td><td>Buxco</td><td>PLY4211</td><td></td></tr><tr><td>Flow meter&amp;nbsp;</td><td>Porter</td><td>F150</td><td></td></tr><tr><td>Bias flow unit</td><td>AFPS</td><td></td><td></td></tr><tr><td>Baseline Gas Mix</td><td>Airgas</td><td>AIZ300</td><td>Compressed Air</td></tr><tr><td>Hypoxic Gas Mix</td><td>Airgas</td><td>X03NI72C2000189</td><td>10% Oxygen, balance nitrogen</td></tr><tr><td>Oxygen Meter</td><td>Teledyne</td><td>AX-300</td><td></td></tr></tbody></table>

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.

Los experimentos iniciales, basados ​​en datos históricos, se realizaron utilizando longitudes de ciclo 1 min. Sobre la base de las calibraciones posteriores realizadas en el Paso 2 anterior, se determinó que el nivel de O 2 en la cámara era 13% en 1 min de lavado post-hipoxia y, que tomó un tiempo similar para volver a la línea de base 21%. Sin embargo, un ciclo de 2 min era suficiente para lograr tanto 10% de oxígeno y un retorno a 21% durante el ciclo de &quot;línea base&quot;. Posteriormente, s...

Watch this Scientific Journal Video about Delivery of In Vivo Acute Intermittent Hypoxia in Neonatal Rodents to Prime Subventricular Zone-derived Neural Progenitor Cell Cultures at JoVE.com

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

En este artículo se describe la metodología para la administración de cortos períodos de hipoxia intermitente para postnatales días 1-8 de ratón o rata crías. Este enfoque suscita efectivamente un nivel de tejido robusto &quot;efecto priming&quot; en células progenitoras neurales cultivadas que se cosechan en los 30 minutos de exposición a la hipoxia.

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 ...

delivery-vivo-acute-intermittent-hypoxia-neonatal-rodents-to-prime

Research

JoVE Journal

Developmental Biology

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

7.2K vistas.  University of Florida.  En este artículo se describe la metodología para la administración de cortos períodos de hipoxia intermitente para postnatales días 1-8 de ratón o rata crías. Este enfoque suscita efectivamente un nivel de tejido robusto "efecto priming" en células progenitoras neurales cultivadas que se cosechan en los 30 minutos de exposición a la hipoxia. 

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

Intraventricular Transplantation of Engineered Neuronal Precursors for In Vivo Neuroarchitecture Studies

Gene control of neuronal cytoarchitecture is currently the subject of intensive investigation. Described here is a simple method developed to study in vivo gene control of neocortical projection neuron morphology. This method is based on (1) in vitro lentiviral engineering of neuronal precursors as "test" and "control" cells, (2) their co-transplantation into wild-type brains, and (3) paired morphometric evaluation of their neuronal derivatives. Specifically, E12.5 pallial precursors from panneuronal, genetically labeled donors, are employed for this purpose. They are engineered to take advantage of selected promoters and tetON/OFF technology, and they are free-hand transplanted into neonatal lateral ventricles. Later, upon immunofluorescence profiling of recipient brains, silhouettes of transplanted neurons are fed into NeurphologyJ open source software, their morphometric parameters are extracted, and average length and branching index are calculated. Compared to other methods, this one offers three main advantages: it permits achieving of fine control of transgene expression at affordable costs, it only requires basic surgical skills, and it provides statistically reliable results upon analysis of a limited number of animals. Because of its design, however, it is not adequate to address non cell-autonomous control of neuroarchitecture. Moreover, it should be preferably used to investigate neurite morphology control after completion of neuronal migration. In its present formulation, this method is exquisitely tuned to investigate gene control of glutamatergic neocortical neuron architecture. Taking advantage of transgenic lines expressing EGFP in other specific neural cell types, it can be re-purposed to address gene control of their architecture.

Based on in vitro lentiviral engineering of neuronal precursors, their co-transplantation into wild-type brains and paired morphometric evaluation of "test" and "control" derivatives, this method allows accurate modeling of in vivo gene control of neocortical neuron morphology in a simple and affordable way.

Trasplante Intraventricular de Precursores Neuronales Deingidos para Estudios de Neuroarquitectura In Vivo

Gene control of neuronal cytoarchitecture is currently the subject of intensive investigation. Described here is a simple method developed to study in ...

Investigación

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

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 ...

Neurociencias

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to neuroblasts able to move along the rostral migratory stream (RMS) towards the olfactory bulb (OB). This long-distance migration is required for the subsequent maturation of newborn neurons in the OB, but the molecular mechanisms regulating this process are still unclear. Investigating the signaling pathways controlling neuroblast motility may not only help understand a fundamental step in neurogenesis, but also have therapeutic regenerative potential, given the ability of these neuroblasts to target brain sites affected by injury, stroke, or degeneration.
In this manuscript we describe a detailed protocol for in vivo postnatal electroporation and subsequent time-lapse imaging of neuroblast migration in the mouse RMS. Postnatal electroporation can efficiently transfect SVZ progenitor cells, which in turn generate neuroblasts migrating along the RMS. Using confocal spinning disk time-lapse microscopy on acute brain slice cultures, neuroblast migration can be monitored in an environment closely resembling the in vivo condition. Moreover, neuroblast motility can be tracked and quantitatively analyzed. As an example, we describe how to use in vivo postnatal electroporation of a GFP-expressing plasmid to label and visualize neuroblasts migrating along the RMS. Electroporation of shRNA or CRE recombinase-expressing plasmids in conditional knockout mice employing the LoxP system can also be used to target genes of interest. Pharmacological manipulation of acute brain slice cultures can be performed to investigate the role of different signaling molecules in neuroblast migration. By coupling in vivo electroporation with time-lapse imaging, we hope to understand the molecular mechanisms controlling neuroblast motility and contribute to the development of novel approaches to promote brain repair.

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

Neuroblast migration is a fundamental event in postnatal neurogenesis. We describe a protocol for efficient labeling of neuroblasts by in vivo postnatal electroporation and subsequent visualization of their migration using time-lapse imaging of acute brain slices. We include a description for the quantitative analysis of neuroblast dynamics by video tracking.

In vivo Postnatal electroporación y time-lapse de Neuroblast Migración en rodajas de cerebro de ratón aguda

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to ...

Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best advance translational objectives and uncover new therapeutic strategies for brain injury associated with preterm birth, preclinical models of EoP must include similar mechanisms of prenatal global injury observed in humans and involve multiple components of the maternal-placental-fetal system. Ideally, models should produce a similar spectrum of functional deficits in the mature animal and recapitulate multiple aspects of the pathophysiology. To mimic human systemic placental perfusion defects, placental underperfusion and/or chorioamnionitis associated with pathogen-induced inflammation in early preterm birth, we developed a model of prenatal transient systemic hypoxia-ischemia (TSHI) combined with intra-amniotic lipopolysaccharide (LPS). In pregnant Sprague Dawley rats, TSHI via uterine artery occlusion on embryonic day 18 (E18) induces a graded placental underperfusion defect associated with increasing CNS damage in the fetus. When combined with intra-amniotic LPS injections, placental inflammation is increased and CNS damage is compounded with associated white matter, gait and imaging abnormalities. Prenatal TSHI and TSHI+LPS prenatal insults meet several of the criteria of an EoP model including recapitulating the intrauterine insult, causing loss of neurons, oligodendrocytes and axons, loss of subplate, and functional deficits in adult animals that mimic those observed in children born extremely preterm. Moreover, this model allows for the dissection of inflammation induced by divergent injury types.

Encephalopathy of prematurity encompasses the central nervous system abnormalities associated with injury from preterm birth. This report describes a clinically relevant rat model of in utero transient systemic hypoxia-ischemia and intra-amniotic lipopolysaccharide administration (LPS) that mimics chorioamnionitis, and the related impact of infectious stimuli and placental underperfusion on CNS development.

Modelado Encefalopatía del Prematuro El uso prenatal hipoxia-isquemia con lipopolisacárido intraamniótica en ratas

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best ...

Medicina

Intratracheal Instillation of Stem Cells in Term Neonatal Rats

Prolonged exposure to high concentrations of oxygen leads to inflammation and acute lung injury, which is similar to human bronchopulmonary dysplasia (BPD). In premature infants, BPD is a major complication despite early use of surfactant therapy, optimal ventilation strategies, and noninvasive positive pressure ventilation. Because pulmonary inflammation plays a crucial role in the pathogenesis of BPD, corticosteroid use is one potential treatment to prevent it. Nevertheless, systemic corticosteroid treatment is not usually recommended for preterm infants due to long-term adverse effects. Preclinical studies and human phase I clinical trials demonstrated that use of mesenchymal stromal cells (MSCs) in hyperoxia-induced lung injuries and in preterm infants is safe and feasible. Intratracheal and intravenous MSC transplantation has been shown to protect against neonatal hyperoxic lung injury. Therefore, intratracheal administration of stem cells and combined surfactant and glucocorticoid treatment has emerged as a new strategy to treat newborns with respiratory disorders. The developmental stage of rat lungs at birth is equivalent to that in human lungs at 26&#8722;28 week of gestation. Hence, newborn rats are appropriate for studying intratracheal administration to preterm infants with respiratory distress to evaluate its efficacy. This intratracheal instillation technique is a clinically viable option for delivery of stem cells and drugs into the lungs.

Described is a protocol for performing intratracheal transplantation of mesenchymal stromal cells (MSCs) through intratracheal injection in term neonatal rats. This technique is a clinically viable option for delivery of stem cells and drugs into neonatal rat lungs to evaluate their efficacy.

Instilación intratraqueal de células madre en ratas neonatales a término

Prolonged exposure to high concentrations of oxygen leads to inflammation and acute lung injury, which is similar to human bronchopulmonary dysplasia ...

Intravenous Injections in Neonatal Mice

Intravenous injection is a clinically applicable manner to deliver therapeutics. For adult rodents and larger animals, intravenous injections are technically feasible and routine. However, some mouse models can have early onset of disease with a rapid progression that makes administration of potential therapies difficult. The temporal (or facial) vein is just anterior to the ear bud in mice and is clearly visible for the first two days after birth on either side of the head using a dissecting microscope. During this window, the temporal vein can be injected with volumes up to 50 &#956;l. The injection is safe and well tolerated by both the pups and the dams. A typical injection procedure is completed within 1-2 min, after which the pup is returned to the home cage. By the third postnatal day the vein is difficult to visualize and the injection procedure becomes technically unreliable. This technique has been used for delivery of adeno-associated virus (AAV) vectors, which in turn can provide almost body-wide, stable transgene expression for the life of the animal depending on the viral serotype chosen.

Animal models of pediatric disease can experience early onset and aggressive disease progression. Clinically relevant therapy delivery to young mouse models can be technically difficult. This protocol describes a non-invasive intravenous injection method for newborn mice within the first two postnatal days of life.

Las inyecciones intravenosas en ratones recién nacidos

Intravenous injection is a clinically applicable manner to deliver therapeutics. For adult rodents and larger animals, intravenous injections are ...

A Minimally Invasive Method for Intratracheal Instillation of Drugs in Neonatal Rodents to Treat Lung Disease

Treatment of neonatal rodent with drugs instilled directly into the trachea could serve as a valuable tool to study the impact of a locally administered drug. This has direct translational impact because surfactant and drugs are administered locally into the lungs. Though the literature has many publications describing minimally invasive transoral intubation of adult mice and rats in therapeutic experiments, this approach in neonatal rat pups is lacking. The small size of orotracheal region/pharynx in the pups makes visualization of laryngeal lumen (vocal cords) difficult, contributing to the variable success rate of intratracheal drug delivery. We hereby demonstrate effective oral intubation of neonatal rat pup - a technique that is non-traumatic and minimally-invasive, so that it can be used for serial administration of drugs. We used an operating otoscope with an illumination system and a magnifying lens to visualize the tracheal opening of the rat neonates. The drug is then instilled using a 1 mL syringe connected to a pipette tip. The accuracy of the delivery method was demonstrated using Evans blue dye administration. This method is easy to get trained in and could serve as an effective way to instill drugs into trachea. This method could also be used for administration of inoculum or agents to simulate disease conditions in animals and, also, for cell-based treatment strategies for various lung diseases.

This technique of instilling drugs directly into the trachea of neonatal rodents is important in studying the impact of locally administered drugs or biologicals on neonatal lung diseases. Additionally, this method can also be used for inducing lung injury in animal models.

Un método mínimamente invasivo para la instilación intratraqueal de medicamentos en roedores neonatales para tratar la enfermedad pulmonar

Treatment of neonatal rodent with drugs instilled directly into the trachea could serve as a valuable tool to study the impact of a locally administered ...

"Ordeño cerebral" de ratas para el aislamiento de células progenitoras de oligodendrocitos y tronco neural

The "Brain Milking" Method for the Isolation of Neural Stem Cells and Oligodendrocyte Progenitor Cells from Live Rats

Tissue-specific neural stem cells (NSCs) remain active in the mammalian postnatal brain. They reside in specialized niches, where they generate new neurons and glia. One such niche is the subependymal zone (SEZ; also called the ventricular-subventricular zone), which is located across the lateral walls of the lateral ventricles, adjacent to the ependymal cell layer. Oligodendrocyte progenitor cells (OPCs) are abundantly distributed throughout the central nervous system, constituting a pool of proliferative progenitor cells that can generate oligodendrocytes.
Both NSCs and OPCs exhibit self-renewal potential and quiescence/activation cycles. Due to their location, the isolation and experimental investigation of these cells is performed postmortem. Here, we describe in detail &#34;brain milking&#34;, a method for the isolation of NSCs and OPCs, amongst other cells, from live animals. This is a two-step protocol designed for use in rodents and tested in rats. First, cells are &#34;released&#34; from the tissue via stereotaxic intracerebroventricular (i.c.v.) injection of a &#34;release cocktail&#34;. The main components are neuraminidase, which targets ependymal cells and induces ventricular wall denudation, an integrin-&#946;1-blocking antibody, and fibroblast growth factor-2. At a second &#34;collection&#34; step, liquid biopsies of cerebrospinal fluid are performed from the cisterna magna, in anesthetized rats without the need of an incision.
Results presented here show that isolated cells retain their endogenous profile and that NSCs of the SEZ preserve their quiescence. The denudation of the ependymal layer is restricted to the anatomical level of injection and the protocol (release and collection) is tolerated well&#160; by the animals. This novel approach paves the way for performing longitudinal studies of endogenous neurogenesis and gliogenesis in experimental animals.

Autor destacado: Recolección de células madre neurales y progenitoras de animales vivos utilizando un novedoso protocolo de ordeño cerebral 

A method for the isolation of neural stem cells and oligodendrocyte progenitor cells from the brains of live rats is presented here in experimental detail. It allows multiple collections of these cells from the same animals without compromising their well-being.

El método de "ordeño cerebral" para el aislamiento de células madre neurales y células progenitoras de oligodendrocitos de ratas vivas

Tissue-specific neural stem cells (NSCs) remain active in the mammalian postnatal brain. They reside in specialized niches, where they generate new ...

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Un método mínimamente invasivo para la instilación intratraqueal de medicamentos en roedores neonatales para tratar la enfermedad pulmonar

El método de "ordeño cerebral" para el aislamiento de células madre neurales y células progenitoras de oligodendrocitos de ratas vivas

El método de "ordeño cerebral" para el aislamiento de células madre neurales y células progenitoras de oligodendrocitos de ratas vivas