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

NOTA: Todos os procedimentos com animais neste protocolo são realizadas com a aprovação da University of Florida Animal Care Institucional e Comitê de Uso (IACUC) e estão em conformidade com o &quot;Guia para o Cuidado e Uso de Animais de Laboratório &#39;. 1. Básica Experimental Set up antes da administração intermitente hipóxia <ol><li> Expor filhotes 12 a diferentes misturas de gás usando uma de corpo inteiro câmaras rato pletismógrafo em muito da mesma forma que roedores adultos para outros fins experimentais 5,13,14. As câmaras têm um diâmetro de 4 polegadas (volume = 450 ml), que é de tamanho suficiente para acomodar 3-4 filhotes de rato ou 1-2 crias de rato. Aqui, os protocolos de AIH são entregues aos recém-nascidos com idade pós-natal 1-8 dia (P1-P8). </li><li> Use tanques de gás de ar comprimido para entregar a 21% do gás &quot;linha de base&quot;, que mantém a oxigenação câmara em 21%. Para alcançar o &quot;hipóxia relativa&quot;, use um / 90% tanque de gás nitrogênio de 10% de oxigênio. NÃOE: tubos de plástico (3/16 &quot;de diâmetro) conecta os tanques de gás de um medidor de caudal. Assim, a entrada para o medidor de fluxo é composto por um tubo para cada linha de base do fluxo de ar e hipóxia. </li><li> Configurar o tubo de gás como se segue. <ol><li> Executar tubo de plástico (diâmetro de 1/8 &quot;) a partir do medidor de fluxo para um regulador de fluxo de polarização, o que permite que os fluxos de 1 L / min para cada câmara (ou seja, 4 litros / min, se são usadas câmaras 4). </li><li> Executar tubo de plástico (diâmetro de 1/8 &quot;) a partir do regulador de fluxo de polarização para as câmaras de pletismografia. O fluxo de ar a 1 L / min, para cada uma das câmaras é um caudal predeterminado em que a troca gasosa é considerada não-indução de stress para os animais, com base em observações comportamentais e fisiológicas. </li></ol></li><li> Vede todas as ligações não utilizadas de e para a unidade de fluxo de polarização e de todas as aberturas não utilizadas para as câmaras com tampas de modo que nenhum fluxo de gás escapa a outras câmaras não em uso, ou para fora das câmaras da pletismografia durante o protocolo. </li><li> Coloque a câmara (s) Em uma incubadora antes de colocar os animais na câmara para permitir o equilíbrio a 37 ° C, ou a temperatura do corpo. </li></ol> 2. Calibração de Ciclo Times por hipóxia intermitente <ol><li> Inspecione toda a tubulação, entre tanques de ar e medidor de fluxo, medidor de fluxo e unidade de fluxo de polarização, e fluxo de viés unidade e câmara (s) pletismografia para as conexões adequadas. As conexões são configurados como descrito no Passo 1. Verifique a conexão por uma verificação passo a passo de todas as linhas descritas e fechamentos: por exemplo, verificando linhas não utilizadas fora da unidade de fluxo de polarização e as aberturas de câmara não estiver em uso. </li><li> Ligue um ambiente sensor de O2 com um O 2 metros de mão e, em seguida, conecte o sensor à tampa da câmara pletismografia. </li><li> Abra os dois tanques de gás e anexar um par de hemostats cirúrgicos, de cabo longo isolados com tubos de plástico para prender o fluxo fora de ciclo de gás. Desta forma, apenas um tanque de gás num momento está a fornecer o / min de fluxo de gás de 1 L por câmara. Prenda a mistura de gases de &quot;linha de base&quot;, enquanto a mistura de gás &quot;hipóxica&quot; é entregue à câmara, e vice-versa. </li><li> Grave o tempo requerido para a câmara de O 2 para alcançar os níveis pretendidos de 10% e regresso a 21%. Utilizar os tempos registrados para entrega protocolo subsequente. </li></ol> 3. Administração de hipóxia intermitente aguda <ol><li> Inspecione toda a tubulação e conexões conforme descrito no Passo 2.1. </li><li> Coloque recém-nascidos na câmara (s) pletismografia e abrigar as tampas de câmara pletismografia na base da câmara. Confirmar uma vedação apropriada para o anel de vedação é assim como o fechamento da câmara de quaisquer ligações não utilizadas. Estes passos são críticos para assegurar o fornecimento adequado de misturas de gás. </li><li> Coloque as câmaras que prendem os filhotes a ser tratada no 37 ° C incubadora e feche a porta. Permitir câmara para equilibrar a 37 ° C antes de se iniciar o protocolo. Manter os animais de controlo a 37 ° C no incubatou na sala de ar oxigenação. </li><li> Abra os dois tanques de gás e usar um par de hemostats cirúrgicos, de cabo longo isolados com tubos de plástico para prender o fluxo fora de ciclo de gás. Desta forma, apenas um tanque de gás num momento está a fornecer o / min de fluxo de gás de 1 L por câmara. </li><li> Use um cronômetro de mão para precisamente ciclos de tempo e para indicar a hora de mudar os hemostats entre tubos, resultando, assim, na alternância do fluxo de gás para as câmaras. </li><li> Grave a conclusão de cada ciclo de tratamento usando um log, como o fornecido na Figura 1 para marcar todas as etapas do 2-step, 20 protocolo de ciclo. </li><li> Monitorar a temperatura da incubadora em cada mudança de ciclo para assegurar animais são mantidos no intervalo designado (33-35 ° C, que é a configuração na incubadora que resulta em 37 ° C). </li><li> Acompanhar de perto a atividade filhote ao longo do protocolo para garantir que os animais tolerar ciclos sem desgaste visível, como vocalizações, alteração do nível deatividade membro ou de rolamento. </li></ol> 4. Isolamento e Cultura de Células tronco / progenitoras da zona subventricular NOTA: A alteração na formação neurosphere seguinte AIH comparados aos controles é um exemplo de um desfecho que demonstra a eficácia deste protocolo para provocar mudanças Tecido e de nível celular. <ol><li> Para isolar neurospheres formando células, remova ratinho ou um rato filhotes da câmara imediatamente após AIH está concluída. Sacrificar os animais de acordo com protocolos IACUC dentro de 30 min de cessação do protocolo. </li><li> Colher o tecido SVZ, coloque na cultura neurosphere e conduzir passagem padrão e expansão da neurospheres derivativos, conforme publicado anteriormente em métodos capítulos 15,16 e um protocolo de vídeo separada 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...

O objetivo deste método é entregar reprodutível e eficaz ataques intermitentes de oxigênio sistêmico ambiente reduzido para roedores neonatais. A justificativa para o uso de hipóxia intermitente (IH) para manipular a biologia de células-tronco origina de em experimentos de cultura de células in vitro em que O 2 conteúdo do meio de crescimento é alterada. Especificamente, quando comparadas com as condições &quot;standard&quot; de 20% de O2, cultura prolongada de células estaminais / progenitoras de células em populações de células 3% de O2 resulta num aumento da proliferação, diminuição e aumento da apoptose neuronal 1,2 rendimento. Este grupo tem uma experiência significativa com a administração de IH sistêmica, e já realizou extensos estudos sobre o papel do IH na plasticidade respiratória 3-7. Este trabalho, ea recente descoberta de que IH crônica aumentou a neurogênese no roedor CNS 10/08, constitui a base para a exploração do aguda in vivohipóxia como um estímulo pré-condicionamento (ie., antes da colheita de tecidos) sobre a cultura subsequente de células estaminais / progenitoras neurais (NPCs) 11. Notavelmente, quando as crias de rato foram expostos a uma breve (&lt;1 hora) período de hipoxia aguda intermitente (AIH), as células que foram colhidas a partir da zona subventricular (SVZ) tinha aumentado significativamente a capacidade de expansão como neuroesferas ou células em monocamadas aderentes. O protocolo AIH também foi associado com o aumento da expressão de um fator de transcrição &quot;destino neuronal&quot; (Pax6). Deste modo, in vivo protocolos AIH pode proporcionar um meio para &quot;Prime&quot; NPCs antes da cultura. Por exemplo, os pedidos de esta abordagem poderia incluir a expansão das populações de células antes do transplante para o sistema nervoso central lesionado, ou simplesmente aumentando a diferenciação neuronal de células cultivadas antes de experiências in vitro. Além disso, porque esta é uma entrega sistémica, qualquerórgão, tecido ou célula é um candidato para o estudo semelhante. Portanto, o protocolo é como escrito potencialmente aplicáveis ​​a uma ampla gama de estudos sobre a manipulação de oxigénio intermitente em pequenos mamíferos. Existem algumas vantagens para esta abordagem. Em outro trabalho publicado, recém-nascidos foram tratados como uma ninhada com a barragem em câmaras hipobáricas, que permite a administração crónica, menos manuseio antes do tratamento, e manteve contato materno durante o tratamento 9. A abordagem actual ultrapassa tratamentos repetidos para uma fêmea de reprodução, ou o uso de uma barragem diferente para cada experimento. Este protocolo também permite o estudo de recém-nascidos pareados por ninhada e da mesma idade precisos. Os dados representativos demonstrar outra força fundamental deste protocolo, ou seja, a rapidez com que AIH, como entregue, provoca uma resposta biológica poderosa e consistente em biologia de células-tronco neurais. Isto estabelece um precedente para esse protocolo para provocar biologi Tecido e-nível celularcal mudanças que alteram a biologia celular. Este relatório irá delinear os procedimentos detalhados utilizados para expor os filhotes de roedores a AIH, bem como a análise da população de células SVZ cultivadas como neurospheres.

<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.

Os experimentos iniciais, com base em dados históricos, foram conduzidos utilizando comprimentos de ciclo 1 min. Com base nos calibrações subsequentes realizados no Passo 2 acima, determinou-se que o nível de O2 na câmara era de 13% em 1 min de lavagem pós-hipoxia e, que teve um tempo semelhante para voltar à linha de base de 21%. No entanto, um ciclo de 2 minutos foi suficiente tanto para alcançar 10% de oxigénio e um retorno a 21% durante o ciclo de &quot;linha de base&quot;. Subsequentemente, os c...

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

Este artigo descreve a metodologia para a administração de curtos períodos de hipóxia intermitente para pós-natal 1-8 dia ratinho ou um rato filhotes. Esta abordagem suscita efectivamente um nível do tecido robusto &quot;efeito priming&quot; sobre as células progenitoras neurais em cultura que são colhidas no prazo de 30 min de exposição hipóxia.

Entrega de<em&gt; In Vivo</em&gt; Aguda intermitente hipóxia em Neonatal Roedores ao primeiro-subventricular Zona derivados de culturas de células progenitoras neurais

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

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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 visualizações.  University of Florida.  Este artigo descreve a metodologia para a administração de curtos períodos de hipóxia intermitente para pós-natal 1-8 dia ratinho ou um rato filhotes. Esta abordagem suscita efectivamente um nível do tecido robusto "efeito priming" sobre as células progenitoras neurais em cultura que são colhidas no prazo de 30 min de exposição hipóxia. 

Vídeo: 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.

Transplante intraventricular de precursores neuronais projetados para estudos de Neuroarquitetura in vivo

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

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.

O Modelo de encefalopatia hipóxico isquêmica Perinatal da Isquemia

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

Biologia

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

Eletroporação Zona Neonatal subventricular

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

Neurociência

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 Eletroporação pós-natal e tempo-lapso de neuroblasto Migração na aguda fatias do cérebro do rato

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.

Modelagem Encefalopatia da Prematuridade Usando pré-natal hipóxia-isquemia com Lipopolysaccharide intra-amniótica em Ratos

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.

Instilação intratraqueal de células-tronco em ratos neonatais de termo

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.

Injeções intravenosas em Neonatal Ratos

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.

Um método minimamente invasivo para a instilação intratraqueal de drogas em roedores neonatais para tratar doenças pulmonares

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 ...

"Ordenha cerebral" de rato para isolamento de células-tronco neurais e oligodendrócitos

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 em destaque: Coletando células-tronco neurais e progenitoras de animais vivos usando um novo protocolo de ordenha 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.

Método "Brain Milking" para o Isolamento de Células-Tronco Neurais e Células Progenitoras de Oligodendrócitos de Ratos Vivos

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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Um método minimamente invasivo para a instilação intratraqueal de drogas em roedores neonatais para tratar doenças pulmonares

Método "Brain Milking" para o Isolamento de Células-Tronco Neurais e Células Progenitoras de Oligodendrócitos de Ratos Vivos

Método "Brain Milking" para o Isolamento de Células-Tronco Neurais e Células Progenitoras de Oligodendrócitos de Ratos Vivos