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

HINWEIS: Alle tierischen Verfahren in diesem Protokoll sind mit Zustimmung der University of Florida Institutional Animal Care und Verwenden Committee (IACUC) durchgeführt und sind in Übereinstimmung mit dem Leitfaden für die Pflege und Verwendung von Labortieren. 1. Grundversuchsaufbau Bevor intermittierende Hypoxie Verwaltung <ol><li> Expose Welpen 12 zu den verschiedenen Gasmischungen unter Verwendung eines Ganzkörper-Maus Plethysmographen Kammern in der gleichen Weise wie erwachsenen Nagetieren für andere experimentelle Zwecke 5,13,14. Die Kammern haben einen Durchmesser von 4 Inch (Volumen = 450 ml), die eine ausreichende Größe ist, um 3-4 Maus Welpen oder 1-2 Rattenjungen zubringen. Hier werden AIH Protokolle Neugeborenen im Alter von postnatalen Tag 1-8 (P1-P8) geliefert. </li><li> Verwenden Sie Druckluft Gastanks, die 21% &quot;Baseline&quot; Gas, das Sauerstoffkammer mit 21% unterhält zu liefern. Um &quot;relative Hypoxie&quot; zu erzielen, verwenden Sie ein% Sauerstoff 10/90% Stickstoffgastank. NICHTE: Kunststoffschlauch (3/16 &quot;Durchmesser) verbindet die Gastanks mit einem Durchflussmesser. Somit wird der Eingang zu dem Durchflussmesser besteht aus jeweils zur Grundlinie und Hypoxie Luftstrom einen Röhre. </li><li> Konfigurieren Sie den Gasschlauch wie folgt. <ol><li> Laufen Kunststoffschlauch (1/8 &quot;Durchmesser) von dem Durchflußmesser zu einem Spannungsströmungsregler, der Ströme von 1 l / min ermöglicht, jede Kammer (dh 4 L / min bei 4 Kammern verwendet werden). </li><li> Führen Kunststoffschlauch (1/8 &quot;Durchmesser) von der Bias-Stromregler auf die Plethysmographie Kammern. Luftstrom bei 1 l / min zu jeder Kammer ist eine vorbestimmte Strömungsrate, mit der der Gasaustausch als Nicht verspannungsinduzierenden der Tiere basierend auf physiologische und Verhaltensbeobachtungen können. </li></ol></li><li> Verschließen Sie alle nicht verwendeten Verbindungen zu und von der Bias-Flow-Einheit und alle nicht verwendeten Öffnungen zu den Kammern mit Kappen, damit kein Gasstrom entweicht, um anderen Kammern nicht in Gebrauch ist, oder aus den Plethysmographie Kammern während des Protokolls. </li><li> Setzen Sie die Kammer (n) In einem Inkubator vor der Inbetriebnahme Tiere in der Kammer, um Äquilibrierung auf 37 ° C, oder Körpertemperatur zu ermöglichen. </li></ol> 2. Kalibrierung der Zykluszeiten für intermittierende Hypoxie <ol><li> Prüfen Sie die Verschlauchung zwischen Luftbehälter und Durchflussmesser, Durchflussmesser und Bias-Flow-Einheit und Bias-Flow-Einheit und Plethysmographie Kammer (n) für die richtigen Verbindungen. ZB ungenutzte Leitungen aus dem Bias-Flow-Einheit und keine Raumöffnungen Überprüfung nicht in Gebrauch: Die Anschlüsse sind wie in Schritt 1 beschrieben, Überprüfen Sie die Verbindung durch eine schrittweise Überprüfung aller beschriebenen Linien und Schließungen gesetzt. </li><li> Schließen Sie eine Umgebungs O 2 -Sensor mit einem Handheld-O 2 Meter und bringen Sie dann den Sensor mit dem Plethysmographie Kammerdeckel. </li><li> Öffnen Sie beide Gastanks und bringen ein Paar chirurgische, langstieligen hemostats mit Kunststoffschlauch isoliert, um die Off-Cycle-Gasstrom zu klemmen. Auf diese Weise wird nur ein Gastank zu einer Zeit liefert die 1 l / min Gasfluss pro Kammer. Klemmen Sie den &quot;Baseline&quot; Gasgemisch, während die &quot;Hypoxie&quot; Gasgemisch in die Kammer geliefert wird, und umgekehrt. </li><li> Notieren Sie sich die Zeit für die Kammer O 2 erforderlich, um die Zielwerte von 10% zu erreichen und zurück zu 21%. Nutzen Sie die aufgezeichneten Zeiten für nachfolgende Protokoll Lieferung. </li></ol> 3. Verwaltung von akuter intermittierender Hypoxie <ol><li> Überprüfen Sie alle Schläuche und Anschlüsse, wie in Schritt 2.1 beschrieben. </li><li> Zeigen Neugeborenen in der Plethysmographie Kammer (n) und das Haus des Plethysmographie Kammerdeckel in die Kammer Basis. Bestätigen Sie eine ordnungsgemäße Abdichtung zu der O-Ringdichtung ist, sowie Schließung von nicht genutzten Kammer Verbindungen. Diese Schritte sind entscheidend für die Gewährleistung der entsprechenden Lieferung von Gasgemischen. </li><li> Zeigen die Kammern halten die Welpen in die 37 ° C-Inkubator behandelt werden und die Tür schließen. Ermöglichen Kammer auf 37 ° C vor dem Beginn des Protokolls äquilibrieren. Aufrechtzuerhalten Kontrolltieren bei 37 ° C im INCUBAToder bei Raumluftsauerstoffversorgung. </li><li> Öffnen Sie beide Gastanks und verwenden Sie ein Paar chirurgische, langstieligen hemostats mit Kunststoffschlauch isoliert, um die Off-Cycle-Gasstrom zu klemmen. Auf diese Weise wird nur ein Gastank zu einer Zeit liefert die 1 l / min Gasstrom pro Kammer. </li><li> Verwenden eines Handauslöser auf präzise Zeitzyklen und die Zeit, um die blutstillende Mittel zwischen den Rohren Schalters anzuzeigen, wodurch der Wechsel der Gasströmung zu den Kammern. </li><li> Nehmen Sie den Abschluss eines jeden Zyklus mit einem Behandlungsprotokoll, wie in Abbildung 1 zur Verfügung gestellt, um zu kennzeichnen Sie alle Schritte in der 2-Stufen, 20-Zyklus-Protokoll. </li><li> Überwachen Sie den Inkubator Temperatur in jedem Zyklus ändern, um sicherzustellen Tiere werden an der vorgesehenen Bereich (33 bis 35 ° C, was die Einstellung auf dem Inkubator, in 37 ° C ergibt, ist) gehalten. </li><li> Pup Tätigkeit während des Protokolls genau überwachen, um sicherzustellen, dass die Tiere vertragen Zyklen ohne sichtbare Not wie Laute, veränderten NiveauGliedmaßentätigkeit oder Walzen. </li></ol> 4. Isolierung und Kultur der Stamm- / Vorläuferzellen aus dem Subventrikularzone HINWEIS: Die Veränderung der Neurosphäre Bildung nach AIH Vergleich zu Kontrollen ist ein Beispiel für einen Endpunkt, die die Wirksamkeit des Protokolls zeigt den Gewebe- und Zellebene Veränderungen hervorzurufen. <ol><li> Um Neurosphären bildenden Zellen zu isolieren, zu entfernen Maus oder Ratte Welpen aus der Kammer unmittelbar nach AIH ist abgeschlossen. Opfern die Tiere nach IACUC Protokolle innerhalb von 30 Minuten von Protokoll-Terminierung. </li><li> Ernten Sie die SVZ Gewebe, legen Sie in die Neurosphärenkultur und Verhalten Standarddurchgang und Expansion von derivativen Neurosphären, wie zuvor im Verfahren Kapiteln 15,16 und einem separaten Video-Protokoll 8 veröffentlicht. </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...

Das Ziel dieser Methode ist es, reproduzierbar und effizient zu liefern intermittierende Anfälle von systemischen abgesenkt Umgebungssauerstoff zu neonatalen Nagetiere. Der Grund für die Verwendung intermittierender Hypoxie (IH) Stammzellen biology manipulieren stammt von in vitro Zellkulturexperimente, in denen O 2 -Gehalt des Wachstumsmediums verändert wird. Insbesondere im Vergleich zu &quot;normalen&quot; Bedingungen von 20% O 2, verlängerte Kultur von Stammzellen / Vorläuferzellpopulationen Zellen in 3% O 2 führt zu einer erhöhten Proliferation, Apoptose verringert und erhöhte neuronale Ausbeute 1,2. Diese Gruppe hat umfangreiche Erfahrungen mit der Verabreichung von systemischen IH und hat umfangreiche Studien über die Rolle der IH in Atem Plastizität 3-7 durchgeführt. Diese Arbeit und die jüngste Entdeckung, dass chronische IH erhöhte Neurogenese im Nagetier CNS 8-10, bildet die Grundlage für die Erforschung der akuten in vivoHypoxie als Vorkonditionierung Stimulus (dh., vor der Gewebeernte) auf die anschließende Kultur von neuronalen Stammzellen / Vorläuferzellen (NPC) 11. Bemerkenswert ist, wenn die Maus Jungtiere wurden auf eine kurze (&lt;1 Stunde) Zeit der akute intermittierende Hypoxie (AIH) ausgesetzt sind, Zellen, die aus der Subventrikularzone (SVZ) geerntet wurden, hatten signifikant erhöhte Kapazität für die Expansion als Neurosphären oder anhaftenden Monolayer-Zellen. AIH Protokoll wurde auch mit einer erhöhten Expression eines &quot;neuronalen Schicksals&quot; Transkriptionsfaktor (Pax6) zugeordnet ist. Dementsprechend wird in vivo AIH Protokolle kann ein Mittel zur &quot;prime&quot; NPCs vor der Kultur zu schaffen. Beispielsweise könnten Anwendungen für diesen Ansatz sind die Erweiterung Zellpopulationen vor der Transplantation in das verletzte Zentralnervensystems, oder einfach die Erhöhung der neuronalen Differenzierung von kultivierten Zellen vor der in vitro-Experimente. Ferner kann, da dies eine systemische Verabreichung, jedeOrgan, Gewebe oder Zellen, ist ein Kandidat für ähnliche Studie. Daher ist das Protokoll geschrieben potenziell für eine breite Palette von Studien über intermittierende Sauerstoff Manipulation in kleinen Säugetieren. Es gibt bestimmte Vorteile für diese Vorgehensweise. In anderen veröffentlichten Arbeiten wurden Neugeborenen als ein Wurf mit der Staumauer in Unterdruckkammern, die für die Langzeitanwendung vor der Behandlung ermöglicht, weniger Handling behandelt und gepflegt mütterlichen Kontakt während der Behandlung 9. Der derzeitige Ansatz umgeht wiederholten Behandlungen zu einer Zuchthündin, oder die Verwendung einer anderen Damm für jedes Experiment. Dieses Protokoll erlaubt auch Studium der präzisen Wurf abgestimmt und altersangepassten Neugeborenen. Repräsentative Daten zeigen eine weitere wesentliche Stärke dieses Protokolls, nämlich die Schnelligkeit, mit der AIH, wie geliefert, löst eine leistungsstarke und einheitliche biologische Reaktion in neuronalen Stammzellbiologie. Dies schafft einen Präzedenzfall für dieses Protokoll, um Gewebe- und Zellebene biologi entlockencal Änderungen, die Zellbiologie zu ändern. Dieser Bericht wird die detaillierten Verfahren zur Belichtung von Nagetier Welpen zur AIH sowie die Populationsanalyse der SVZ-Zellen als Neurosphären verwendet gewachsen zu skizzieren.

<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.

Die ersten Versuche, basierend auf historischen Daten, wurden unter Verwendung von 1 min Zykluslängen durchgeführt. Auf der Grundlage der nachfolgenden Kalibrierungen in Schritt 2 durchgeführt wird, wurde bestimmt, dass das O 2 in der Kammer, betrug 13% nach 1 min nach dem Spülen und Hypoxie, sie habe eine ähnliche Zeit, um die 21% Grundlinie zurück. Allerdings war eine 2 Minuten-Zyklus aus, um beides zu erreichen 10% Sauerstoff und eine Rückkehr zu 21% während des &quot;Baseline&quot; Zyklus. Anschli...

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

Dieser Artikel beschreibt die Methodik für die Verwaltung von kurzen Perioden intermittierender Hypoxie zu postnatalen Tag 1-8 Maus oder Ratte Welpen. Dieser Ansatz effektiv löst eine robuste Gewebeniveau &quot;Priming-Effekt&quot; an kultivierten neuralen Vorläuferzellen, die innerhalb von 30 Minuten von Hypoxie Exposition geerntet werden.

Lieferung von<em&gt; In-vivo-</em&gt; Akuter intermittierender Hypoxie in Neonatal Nagetiere in den Prime Subventrikularzone abgeleiteten neuronalen Vorläuferzellkulturen

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 Aufrufe.  University of Florida.  Dieser Artikel beschreibt die Methodik für die Verwaltung von kurzen Perioden intermittierender Hypoxie zu postnatalen Tag 1-8 Maus oder Ratte Welpen. Dieser Ansatz effektiv löst eine robuste Gewebeniveau "Priming-Effekt" an kultivierten neuralen Vorläuferzellen, die innerhalb von 30 Minuten von Hypoxie Exposition geerntet werden. 

Serie: 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.

Intraventrikuläre Transplantation von 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 ...

Forschung

Genetik

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.

Die hypoxische ischämischen Enzephalopathie Model of Perinatal Ischämie

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

Biologie

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

Neonatal Subventrikulärzone Elektroporation

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

Neurowissenschaften

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-Elektroporation und Postnatale Zeitraffer-Imaging Neuroblast von Migration in Maus Akute Hirnschnitten

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

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

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

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

Intracerebroventricular Viral Injektion der Neonatal Maus Gehirn für anhaltenden und verbreiteten Neuronale Transduction

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

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to intracranial delivery, intra-arterial administration of NSCs is less invasive and produces a more diffuse distribution of NSCs within the brain parenchyma. Further, intra-arterial delivery allows the first-pass effect in the brain circulation, lessening the potential for trapping of cells in peripheral organs, such as liver and spleen, a complication associated with peripheral injections. Here, we detail the methodology, in both mice and rats, for delivery of NSCs through the common carotid artery (mouse) or external carotid artery (rat) to the ipsilateral hemisphere after an ischemic stroke. Using GFP-labeled NSCs, we illustrate the widespread distribution achieved throughout the rodent ipsilateral hemisphere at 1 d, 1 week and 4 weeks after postischemic delivery, with a higher density in or near the ischemic injury site. In addition to long-term survival, we show evidence of differentiation of GFP-labeled cells at 4 weeks. The intra-arterial delivery approach described here for NSCs can also be used for administration of therapeutic compounds, and thus has broad applicability to varied CNS injury and disease models across multiple species.

A method for delivering neural stem cells, adaptable for injecting solutions or suspensions, through the common carotid artery (mouse) or external carotid artery (rat) after ischemic stroke is reported. Injected cells are distributed broadly throughout the brain parenchyma and can be detected up to 30 d after delivery.

Intra-arterielle Lieferung von neuronalen Stammzellen an das Ratten- und Maushirn: Anwendung auf zerebrale Ischämie

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to ...

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.

Intratracheale Instillation von Stammzellen bei Term Neonatal Ratten

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

Medizin

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.

Intravenöse Injektionen in neugeborenen Mäusen

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.

Eine minimal-invasive Methode zur intratrachealen Instillation von Medikamenten bei neonatalen Nagetieren zur Behandlung von Lungenerkrankungen

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 ...

Ratten-"Gehirnmelken" für die Isolierung von neuralen Stamm- und Oligodendrozyten-Vorläuferzellen

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 im Rampenlicht: Sammeln von neuralen Stamm- und Vorläuferzellen von lebenden Tieren mit einem neuartigen Gehirnmelkprotokoll 

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.

Die "Brain Milking"-Methode zur Isolierung von neuralen Stammzellen und Oligodendrozyten-Vorläuferzellen aus lebenden Ratten

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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