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

NOTA: Tutte le procedure animali in questo protocollo sono condotte con l&#39;approvazione della University of Florida Istituzionale Animal Care and Use Committee (IACUC) e sono in conformità con la &#39;Guida per la cura e l&#39;uso di animali da laboratorio&#39;. 1. Impostare sperimentale di base in funzione Prima Amministrazione intermittente ipossia <ol><li> Esporre cuccioli 12 alle diverse miscele di gas che utilizzano un corpo intero camere del mouse pletismografo più o meno allo stesso modo di roditori adulti per altri scopi sperimentali 5,13,14. Le camere hanno un diametro di 4 pollici (volume = 450 ml), che è di ampie dimensioni per ospitare 3-4 cuccioli del mouse o 1-2 cuccioli di ratto. Qui, i protocolli di AIH sono consegnati a neonati di età compresa tra 1-8 giorni post-natale (P1-P8). </li><li> Utilizzare serbatoi di gas compresso l&#39;aria di consegnare il gas 21% &quot;di base&quot;, che mantiene l&#39;ossigenazione camera al 21%. Per raggiungere &quot;ipossia relativa&quot;, utilizzare un / 90% serbatoio di gas di azoto 10% di ossigeno. NONE: Tubi flessibili (&quot;diametro 3/16) collega i serbatoi di gas a un flussometro. Pertanto, l&#39;ingresso del flussometro è costituito da un tubo per ciascuna linea di base e ipossia flusso d&#39;aria. </li><li> Configurare il tubo del gas come segue. <ol><li> Eseguire tubazione di plastica (&quot;diametro 1/8) dal flussometro di un regolatore di flusso di polarizzazione, che consente flussi di 1 L / min per ciascuna camera (cioè, 4 L / min se si utilizzano 4 alloggiamenti). </li><li> Eseguire tubazione di plastica (&quot;diametro 1/8) dal regolatore di flusso polarizzazione alle camere pletismografia. Flusso d&#39;aria a 1 L / min per ciascuna camera è una portata predeterminata in cui lo scambio di gas è considerata non-stressanti agli animali basate su osservazioni fisiologiche e comportamentali. </li></ol></li><li> Chiudere tutte le connessioni inutilizzate da e per l&#39;unità di flusso pregiudizi e tutte le aperture non utilizzate alle camere con tappi in modo che nessun flusso di gas sfugge ad altre stanze non in uso, o fuori delle camere pletismografia durante il protocollo. </li><li> Posizionare la camera (s) In un incubatore prima di mettere animali nella camera per raggiungere l&#39;equilibrio a 37 ° C, o della temperatura corporea. </li></ol> 2. Taratura di tempi di ciclo per ipossia intermittente <ol><li> Controllare tutti i tubi, tra bombole e flussometro, flussometro e unità di flusso pregiudizi, e il flusso di pregiudizi unità e la camera pletismografia (s) per le connessioni appropriate. I collegamenti sono impostati come descritto al punto 1. Verificare la connessione da un controllo graduale di tutte le linee descritte e chiusure: ad esempio, il controllo linee usate fuori dalla unità di flusso di polarizzazione e le aperture di camera non è in uso. </li><li> Collegare un sensore ambientale O 2 con una O 2 metri portatile e quindi collegare il sensore al coperchio della camera pletismografia. </li><li> Aprire entrambi i serbatoi del gas e collegare un paio di chirurgia, emostatici manico lungo isolati con tubi di plastica per bloccare il flusso di fuori-ciclo del gas. In questo modo, un solo serbatoio alla volta è fornire il / flusso del gas min 1 L per ogni camera. Bloccare la miscela di gas &quot;di base&quot;, mentre la miscela di gas &quot;ipossia&quot;, è consegnato alla camera, e viceversa. </li><li> Registrare il tempo necessario per la camera 2 O per raggiungere i livelli obiettivo di 10% e tornare al 21%. Utilizzare i tempi di consegna per il successivo protocollo. </li></ol> 3. La somministrazione di ipossia acuta intermittente <ol><li> Controllare tutti i tubi e le connessioni come descritto al punto 2.1. </li><li> Posizionare neonati nella camera pletismografia (s) e ospitare le palpebre camera pletismografia nella base della camera. Confermare una corretta tenuta della guarnizione O-ring è così come la chiusura di tutti i collegamenti da camera inutilizzate. Questi passaggi sono fondamentali per assicurare la consegna adeguata di miscele di gas. </li><li> Posizionare le camere che tengono i cuccioli da trattare nel 37 ° C incubatore e chiudono la porta. Lasciare camera di equilibrare a 37 ° C prima di iniziare il protocollo. Mantenere animali di controllo a 37 ° C nel incubato in camera d&#39;aria ossigenazione. </li><li> Aprire entrambi i serbatoi di gas e utilizzare una coppia di chirurgia, emostatici manico lungo isolati con tubi di plastica per bloccare il flusso di fuori-ciclo del gas. In questo modo, un solo serbatoio alla volta è fornire il / flusso del gas min 1 L per ogni camera. </li><li> Utilizzare un timer manuale a proprio cicli di tempo e per indicare il momento di cambiare le hemostats tra tubi, con conseguente l&#39;alternanza del flusso di gas verso le camere. </li><li> Registrare il completamento di ogni ciclo utilizzando un registro trattamento come quello fornito in Figura 1 per delimitare tutte le fasi del 2-step, 20 protocollo ciclo. </li><li> Monitorare la temperatura dell&#39;incubatrice ad ogni cambio ciclo per assicurare gli animali sono mantenuti al raggio designato (33-35 ° C, che è l&#39;impostazione sull&#39;incubatrice che si traduce in 37 ° C). </li><li> Seguire da vicino l&#39;attività cucciolo tutto il protocollo per garantire che gli animali tollerano cicli senza angoscia visibile come vocalizzi, alterato livello diattività arto o di rotolamento. </li></ol> 4. Isolamento e la coltura di cellule staminali / progenitrici dalla zona subventricolare NOTA: Il cambiamento di formazione neurosfere seguito EAI rispetto ai controlli è un esempio di un endpoint che dimostra l&#39;efficacia di questo protocollo per suscitare cambiamenti tessutale e a livello cellulare. <ol><li> Per isolare neurosfere formano cellule, rimuovere topo o ratto cuccioli dalla camera subito dopo EAI è completata. Sacrificare gli animali secondo protocolli IACUC entro 30 minuti dal termine del protocollo. </li><li> Raccolto il tessuto SVZ, inserire nella cultura neurosfere e condurre iter standard e l&#39;espansione delle neurosfere derivate, come precedentemente pubblicato su metodi capitoli 15,16 e un protocollo video separata 8. </li></ol>

<ol><li>Studer, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enhanced+proliferation%2C+survival%2C+and+dopaminergic+differentiation+of+CNS+precursors+in+lowered+oxygen%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Enhanced proliferation, survival, and dopaminergic differentiation of CNS precursors in lowered oxygen.</a> J Neurosci. 20 (19), 7377-7383 (2000).</li>
<li>Chen, H. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Oxygen+tension+regulates+survival+and+fate+of+mouse+central+nervous+system+precursors+at+multiple+levels%5BTitle%5D)+AND+%22Stem+Cells%22%5BJournal%5D)">Oxygen tension regulates survival and fate of mouse central nervous system precursors at multiple levels.</a> Stem Cells. 25 (9), 2291-2301 (2007).</li>
<li>Ling, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chronic+intermittent+hypoxia+elicits+serotonin-dependent+plasticity+in+the+central+neural+control+of+breathing%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Chronic intermittent hypoxia elicits serotonin-dependent plasticity in the central neural control of breathing.</a> J Neurosci. 21 (14), 5381-5388 (2001).</li>
<li>Mitchell, G. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Invited+review%3A+Intermittent+hypoxia+and+respiratory+plasticity%5BTitle%5D)+AND+%22J+Appl+Physiol%22%5BJournal%5D)">Invited review: Intermittent hypoxia and respiratory plasticity.</a> J Appl Physiol. 90 (6), 2466-2475 (2001).</li>
<li>Fuller, D. D., Zabka, A. G., Baker, T. L., Mitchell, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Phrenic+long-term+facilitation+requires+5-HT+receptor+activation+during+but+not+following+episodic+hypoxia%5BTitle%5D)+AND+%22J+Appl+Physiol%22%5BJournal%5D)">Phrenic long-term facilitation requires 5-HT receptor activation during but not following episodic hypoxia.</a> J Appl Physiol. 90 (5), 2001-2006 (2001).</li>
<li>Fuller, D. D., Johnson, S. M., Olson, E. B., Mitchell, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Synaptic+pathways+to+phrenic+motoneurons+are+enhanced+by+chronic+intermittent+hypoxia+after+cervical+spinal+cord+injury%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Synaptic pathways to phrenic motoneurons are enhanced by chronic intermittent hypoxia after cervical spinal cord injury.</a> J Neurosci. 23 (7), 2993-3000 (2003).</li>
<li>Baker-Herman, T. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((BDNF+is+necessary+and+sufficient+for+spinal+respiratory+plasticity+following+intermittent+hypoxia%5BTitle%5D)+AND+%22Nat+Neurosci%22%5BJournal%5D)">BDNF is necessary and sufficient for spinal respiratory plasticity following intermittent hypoxia.</a> Nat Neurosci. 7 (1), 48-55 (2004).</li>
<li>Zhu, L. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neurogenesis+in+the+adult+rat+brain+after+intermittent+hypoxia%5BTitle%5D)+AND+%22Brain+Res%22%5BJournal%5D)">Neurogenesis in the adult rat brain after intermittent hypoxia.</a> Brain Res. 1055, 1-6 (2005).</li>
<li>Zhang, J. X., Chen, X. Q., Du, J. Z., Chen, Q. M., Zhu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neonatal+exposure+to+intermittent+hypoxia+enhances+mice+performance+in+water+maze+and+8-arm+radial+maze+tasks%5BTitle%5D)+AND+%22Journal+of+neurobiology%22%5BJournal%5D)">Neonatal exposure to intermittent hypoxia enhances mice performance in water maze and 8-arm radial maze tasks.</a> Journal of neurobiology. 65 (1), 72-84 (2005).</li>
<li>Zhu, L. L., Wu, L. Y., Yew, D. T., Fan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+hypoxia+on+the+proliferation+and+differentiation+of+NSCs%5BTitle%5D)+AND+%22Mol+Neurobiol%22%5BJournal%5D)">Effects of hypoxia on the proliferation and differentiation of NSCs.</a> Mol Neurobiol. 31 (1-3), 231-242 (2005).</li>
<li>Ross, H. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+intermittent+hypoxia+elicits+enhanced+expansion+and+neuronal+differentiation+in+cultured+neural+progenitors%5BTitle%5D)+AND+%22Exp+Neurol%22%5BJournal%5D)">In vivo intermittent hypoxia elicits enhanced expansion and neuronal differentiation in cultured neural progenitors.</a> Exp Neurol. 235 (1), 238-245 (2012).</li>
<li>Fuller, D. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Induced+recovery+of+hypoxic+phrenic+responses+in+adult+rats+exposed+to+hyperoxia+for+the+first+month+of+life%5BTitle%5D)+AND+%22J+Physiol%22%5BJournal%5D)">Induced recovery of hypoxic phrenic responses in adult rats exposed to hyperoxia for the first month of life.</a> J Physiol. 536 (Pt 3), 917-926 (2001).</li>
<li>Fuller, D. D., Fregosi, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fatiguing+contractions+of+tongue+protrudor+and+retractor+muscles%3A+influence+of+systemic+hypoxia%5BTitle%5D)+AND+%22J+Appl+Physiol%22%5BJournal%5D)">Fatiguing contractions of tongue protrudor and retractor muscles: influence of systemic hypoxia.</a> J Appl Physiol. 88 (6), 2123-2130 (2000).</li>
<li>Baker, T. L., Fuller, D. D., Zabka, A. G., Mitchell, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Respiratory+plasticity%3A+differential+actions+of+continuous+and+episodic+hypoxia+and+hypercapnia%5BTitle%5D)+AND+%22Respir+Physiol%22%5BJournal%5D)">Respiratory plasticity: differential actions of continuous and episodic hypoxia and hypercapnia.</a> Respir Physiol. 129 (1-2), 25-35 (2001).</li>
<li>Marshall, G. P. 2nd, et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Production+of+neurospheres+from+CNS+tissue%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Production of neurospheres from CNS tissue.</a> Methods Mol Biol. 438, 135-150 (2008).</li>
<li>Azari, H., Rahman, M., Sharififar, S., Reynolds, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Isolation+and+expansion+of+the+adult+mouse+neural+stem+cells+using+the+neurosphere+assay%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Isolation and expansion of the adult mouse neural stem cells using the neurosphere assay.</a> J Vis Exp. (45), (2010).</li>
</ol>

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...

L&#39;obiettivo di questo metodo è quello di fornire riproducibile ed efficace periodi intermittenti di sistemica di ossigeno ambiente abbassata a roditori neonatali. Il razionale per l&#39;utilizzo di ipossia intermittente (IH) per manipolare biologia delle cellule staminali in vitro proviene da esperimenti di coltura cellulare in cui tenore di O 2 i mezzi di crescita è alterata. In particolare, rispetto alle condizioni &quot;standard&quot; del 20% O 2, cultura estesa staminali / progenitrici delle cellule popolazioni cellulari in 3% O 2 risultati in un aumento della proliferazione, diminuita apoptosi neuronale e una maggiore resa 1,2. Questo gruppo ha una significativa esperienza con la somministrazione di IH sistemica, e ha condotto studi approfonditi sul ruolo della IH nella plasticità respiratoria 3-7. Questo lavoro, e la recente scoperta che IH cronica aumentata neurogenesi nei roditori CNS 8-10, costituisce la base per l&#39;esplorazione di acuta in vivoipossia come stimolo precondizionamento (es., prima del raccolto tessuti) sulla successiva coltura di cellule neurali staminali / progenitrici (NPC) 11. Sorprendentemente, quando cuccioli di topo sono stati esposti a un breve periodo di acuta ipossia intermittente (IAC) (&lt;1 ora), cellule che sono state raccolte dalla zona subventricolare (SVZ) era significativo aumento di capacità di espansione come neurosfere o cellule monostrato aderenti. Il protocollo AIH è stato anche associato con una maggiore espressione di un &quot;destino neuronale&quot; fattore di trascrizione (Pax6). Di conseguenza, in vivo protocolli AIH possono fornire un mezzo di &quot;prime&quot; NPC prima cultura. Ad esempio, applicazioni di questo approccio possono includere espansione popolazioni di cellule prima del trapianto nel sistema nervoso centrale feriti, o semplicemente aumentando la differenziazione neuronale di cellule coltivate prima esperimenti in vitro. Inoltre, perché questa è una consegna sistemica, qualsiasiorgano, tessuto o cellula è un candidato per studio simile. Pertanto, il protocollo come scritto è potenzialmente applicabile ad una vasta gamma di studi sulla manipolazione ossigeno intermittente in piccoli mammiferi. Ci sono alcuni vantaggi di questo approccio. In un altro lavoro pubblicato, i neonati sono stati trattati come una cucciolata con la diga in camere ipobariche, che consente la somministrazione cronica, meno manipolazione prima del trattamento, e mantenuto il contatto materno durante il trattamento 9. L&#39;attuale approccio bypassa trattamenti ripetuti ad una femmina di allevamento, o l&#39;uso di una diga differente per ciascun esperimento. Questo protocollo permette anche lo studio di precisi neonati lettiere corrispondenza e di pari età. Dati rappresentativi dimostrano un altro punto di forza di questo protocollo, vale a dire la rapidità con cui epatite autoimmune, come consegnato, provoca una risposta biologica potente e costante in biologia delle cellule staminali neurali. Questo stabilisce un precedente per questo protocollo di suscitare biologi tessutale e-livello cellularemodifiche CAL che alterano biologia cellulare. La relazione illustrerà le procedure dettagliate utilizzate per esporre cuccioli roditori per EAI, nonché l&#39;analisi della popolazione di cellule SVZ cresciuto come neurosfere.

<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.

Gli esperimenti iniziali, basate su dati storici, sono stati condotti utilizzando durata del ciclo 1 min. Sulla base delle successive calibrazioni eseguite nella Fase 2, si è stabilito che il livello di O 2 nella camera era 13% a 1 min lavaggio post-ipossia e, che ha un tempo simile al ritorno alla linea di base 21%. Tuttavia, un ciclo di 2 minuti era sufficiente per realizzare sia il 10% di ossigeno e un ritorno al 21% durante il ciclo di &quot;base&quot;. Successivamente, sono stati utilizzati durata del 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

Questo articolo descrive la metodologia per la somministrazione di brevi periodi di ipossia intermittente per postnatali giorno 1-8 di topo o ratto cuccioli. Questo approccio suscita effettivamente un livello del tessuto robusto &quot;effetto priming&quot; su colture di cellule progenitrici neurali che vengono raccolte entro 30 minuti dall&#39;esposizione ipossia.

Consegna di<em&gt; In Vivo</em&gt; Acuta intermittente ipossia in Neonatale Roditore al Primo subventricolare Zone-derivati ​​colture cellulari progenitrici neurali

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 Views.  University of Florida.  Questo articolo descrive la metodologia per la somministrazione di brevi periodi di ipossia intermittente per postnatali giorno 1-8 di topo o ratto cuccioli. Questo approccio suscita effettivamente un livello del tessuto robusto "effetto priming" su colture di cellule progenitrici neurali che vengono raccolte entro 30 minuti dall'esposizione ipossia. 

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.

Trapianto intraventricolare di precursori neuronali ingegnerizzati per studi sulla neuroarchitettura In Vivo

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

Ricerca

Genetica

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.

Il modello di encefalopatia ipossico ischemica di ischemia perinatale

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

Neonatale Elettroporazione zona subventricolare

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

Neuroscienze

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 postnatale elettroporazione e Time-lapse imaging di migrazione neuroblasti in mouse acuta Fette cervello

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.

Modellazione Encefalopatia di prematurità Utilizzando prenatale ipossia-ischemia con Lipopolisaccaride intra-amniotico in ratti

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

Medicina

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.

Consegna intra-arteriosa di cellule staminali neurali al cervello di ratto e topo: applicazione all'ischemia cerebrale

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.

Instillazione intratracheale delle cellule staminali nei " ratti neooatali

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.

Le iniezioni per via endovenosa nel topo neonatale

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 metodo minimamente invasivo per l'instillazione intratracheale di farmaci nei roditori neonatali per il trattamento delle malattie polmonari

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 ...

"Mungitura cerebrale" di ratto per l'isolamento delle cellule progenitrici del tronco neurale e degli oligodendrociti

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.

Autore in primo piano: Raccolta di cellule staminali neurali e progenitrici da animali vivi utilizzando un nuovo protocollo di mungitura cerebrale 

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.

Il metodo di "mungitura del cervello" per l'isolamento di cellule staminali neurali e cellule progenitrici di oligodendrociti da ratti vivi

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

Consegna di

Riepilogo

Esplora Altri Video