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

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

NOT: Bu protokol tüm hayvan prosedürleri Florida Kurumsal Hayvan Bakımı ve Kullanımı Komitesi (IACUC) Üniversitesi onayı ile gerçekleştirilen ve &#39;Laboratuvar Hayvanları Bakım ve Kullanım Kılavuzu&#39; ile uyumlu vardır. 1. Temel Deney Seti yukarı Aralıklı Hipoksi İdaresi Öncesi <ol><li> Diğer deneysel amaçlar 5,13,14 yetişkin kemirgenler kadar aynı şekilde bir tüm vücut fare Pletismograf odaları kullanarak farklı gaz karışımları için yavrular 12 Açığa. Odaları 3-4 fare yavrular ya da 1-2 sıçan yavrular karşılamak için yeterli büyüklükte bir 4 inç çapı (hacim = 450 ml) var. Burada, AIH protokolleri doğum sonrası gün 1-8 (P1-P8) yaş yenidoğanlarda teslim edilir. </li><li> % 21 kamara oksijenasyonu tutar% 21 &quot;taban&quot; gaz teslim basınçlı hava gaz tankları kullanın. &quot;Göreli hipoksi&quot; elde etmek için,% 10 oksijen /% 90 azot gazı tankı kullanın. DEĞİLE: Plastik boru (3/16 &quot;çapında) bir akış ölçere gaz tankları bağlanır. Bu nedenle, akış ölçere girişi bir tüp taban çizgisi ile hipoksi hava akışı için, her oluşur. </li><li> Aşağıdaki gibi gaz boru yapılandırın. <ol><li> Her odaya 1 L / dk akışını sağlayan bir önyargı akış regülatörü, akış ölçer plastik boru (1/8 &quot;çap) Run (yani 4 L / dk 4 odacıklı kullanılırsa). </li><li> Pletismografi odalarına önyargı akış düzenleyici plastik boru (1/8 &quot;çap) çalıştırın. Hava akımı, 1 L / saat, her bölmeye dakika gaz alışverişi olmayan stres yapıcı fizyolojik ve davranış gözlemlere dayanarak hayvanlara olduğu kabul edildiği bir önceden tespit edilmiş bir akış hızıdır. </li></ol></li><li> Hiçbir gaz akış protokolü sırasında kullanılmadığı, ya da pletismografi odaları dışında diğer odalarına kaçar ve böylece kapakları odalarına ve önyargı akış biriminden kullanılmayan tüm bağlantıları ve tüm kullanılmayan açıklıkları kapatılmalıdır. </li><li> (Odasına yerleştirin ler) 37 ° C&#39;ye kadar dengeye veya vücut ısısı sağlamak için bölme hayvanların koyarak önce bir inkübatör içine. </li></ol> Aralıklı Hipoksi için Döngüsü Times 2. Kalibrasyon <ol><li> Hava tankları ve akış ölçer arasındaki tüm hortumunu kontrol edin doğru bağlantıları için sayaç ve önyargı akış ünitesi ve önyargı akış ünitesi ve pletismografisi odasını (ler) akar. Örneğin, kullanımda olmayan kullanılmayan önyargı akış biriminin dışına hatları ve herhangi bir odacık açıklıkları kontrol: Tüm açıklanan hatları ve kapanışları bir adım adım çekle bağlantısını kontrol edin Adım 1&#39;de açıklandığı gibi bağlantı kurulmaktadır. </li><li> Bir el O 2 metre ile ortam O 2 sensörünü bağlayın ve sonra pletismografisi odası kapak sensörü takın. </li><li> Her iki gaz tankları açın ve gaz off-döngüsü akışını kelepçe plastik boru ile izole cerrahi, uzun saplı hemostat bir çift takın. Bu şekilde, bir seferde sadece bir gaz tankeri odasının başına 1 L / dk gaz akışını iletiyor. &quot;Hipoksik&quot; gaz karışımı tersi odasına teslim ve ise &quot;taban&quot; gaz karışımı Kelepçe. </li><li> % 10 hedef düzeylere ulaşması ve% 21 geri dönmek için hazne O 2 için gerekli süreyi kaydedin. Sonraki protokol teslimat için kaydedilen süreleri yararlanın. </li></ol> Akut Aralıklı Hipoksi 3. İdare <ol><li> Adım 2.1 anlatıldığı gibi tüm boru ve bağlantıları kontrol edin. </li><li> Pletismografisi odasının (ler) yenidoğan yerleştirin ve oda üssü haline pletismografisi odası kapaklarını ev. Kullanılmayan odası bağlantılarının uygun bir O-ring conta ile mühür olduğu gibi kapatma onaylayın. Bu adımlar, gaz karışımları uygun teslimat sağlanması için kritik öneme sahiptir. </li><li> Yavrular tutan odaları 37 ° C inkübatör içine tedavi edilmesi yerleştirin ve kapağını kapatın. Kamara önce protokolü başlamadan 37 ° C gelmesini sağlayınız. Incubat 37 ° C &#39;de kontrol hayvanları korumakveya oda havası oksijenasyonu de. </li><li> Her iki gaz tankları açın ve gaz off-döngüsü akışını kelepçe plastik boru ile izole cerrahi, uzun saplı hemostat bir çift kullanın. Bu şekilde, bir seferde sadece bir gaz tankeri odasının başına 1 L / dk gaz akışının sağlamaktadır. </li><li> Hassas bir zaman döngü elde tutulan bir zamanlayıcı kullanarak ve böylece odalarına gaz akışının münavebe ile sonuçlanan, borular arasındaki hemostat geçiş zamanı gösterir. </li><li> Örneğin 2 aşamalı, 20 çevrim protokolünde tüm adımları kapalı işaretlemek için Şekil 1&#39;de verilen biri olarak tedavi günlüğünü kullanarak her döngünün tamamlanmasını kaydedin. </li><li> Hayvanlar belirlenen aralığın (37 ° C ile sonuçlanan inkübatör ayarı 33-35 ° C) muhafaza edilir sağlamak için her döngü değişikliğinde inkübatör sıcaklığını izleyin. </li><li> Yakından hayvanlar gibi sesler çıkarma, değişmiş seviyesi gibi görünen sıkıntı olmadan çevrimleri tahammül emin olmak için protokol boyunca yavru etkinliğini izlemekuzuv faaliyet veya yuvarlanma. </li></ol> Subventricular Zone Kök / Progenitör Hücreler 4. İzolasyon ve Kültür NOT: kontrollere göre AIH aşağıdaki neurosphere oluşumunda değişiklik doku ve hücresel düzey değişiklikleri ortaya çıkarma, bu protokolün etkinliğini gösteren bir bitiş noktası bir örnektir. <ol><li> Hücreleri oluşturan neurospheres izole etmek için, AIH tamamlandıktan hemen sonra odadan fare veya sıçan yavrular kaldırın. Protokol fesih 30 dk içinde IACUC protokollerine göre hayvanların Kurban. </li><li> Daha önce yöntemler bölümlerde 15,16 ve ayrı bir video protokolü 8 yayımlanan, SVZ doku hasat neurosphere kültürü yerleştirmek ve standart geçit ve türev neurospheres genişlemesini yürütmek. </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...

Bu yöntemin amacı, yeniden üretilebilir ve etkili bir yenidoğan kemirgenlere sistemik alçaltılmış havada bulunan oksijen aralıklı nöbetleri sunmaktır. Kök hücre biyolojisi işlemek için kesintili hipoksi (İH) kullanımı için gerekçe büyüme ortamı O 2 içeriği değiştirilmiş olduğu in vitro hücre kültürü deneylerinde kaynaklanır. % 20 &quot;standart&quot; koşullar O 2, artan çoğalmasında% 3 O 2 sonuçlarında kök / progenitör hücre popülasyonları hücrelerin uzatılmış kültürü ile karşılaştırıldığında Özellikle, apoptozu azalmış ve nöronal verim 1,2 arttı. Bu grup, sistemik IH yönetimi ile önemli bir deneyime sahiptir ve solunum plastisitede 3-7 IH rolü üzerine kapsamlı çalışmalar yürütmüştür. Bu çalışma, kronik IH kemirgen CNS 8-10 nörogenezisi arttığı son bulgu in vivo akut keşif için temelini oluştururBir önkoşullanma uyarıcı olarak hipoksi (yani., doku hasat öncesinde) nöral kök / progenitör hücrelerin (NPC) 11 sonraki kültürüne. Fare yavrular akut aralıklı hipoksi (AIH) kısa (&lt;1 saat) Dönem maruz kaldılar Dikkate değer, subventriküler bölge (SVZ) hasat edildi hücreler önemli ölçüde neurospheres veya yapışık tek tabaka hücreleri gibi genişleme kapasitesi artmıştır. AİH protokol aynı zamanda bir &quot;nöron kaderi&quot; transkripsiyon faktörü (Pax6) artmış ekspresyonu ile ilişkili bulunmuştur. Buna göre, in vivo AIH protokolleri için &quot;prime&quot; NPC&#39;ler önce kültürüne bir araç sağlayabilir. Örneğin, bu yaklaşım için uygulamalar yaralı merkezi sinir sistemi içine transplantasyondan önce hücre popülasyonlarının genişletilmesi, ya da sadece önceden in vitro deneylere kültürlenmiş hücrelerin nöronal farklılaşma artan içerebilir. Bundan başka, bu sistemik olarak uygulanması, çünkü herhangi birorgan doku veya hücre içindeki çalışma için bir adaydır. Bu nedenle, yazılı olarak protokol küçük memeliler aralıklı oksijen manipülasyon çalışmalarının geniş bir yelpazede potansiyel uygulanabilir. Bu yaklaşımın bazı avantajları vardır. Diğer yayınlanan çalışmada, yenidoğanlar tedaviden önce, kronik dozlama için daha az işleme izin veren hipobarik odacıklarda baraj, bir çöp muamelesi ve tedavisi 9 süresince anne teması muhafaza edildi. Geçerli yaklaşım üreme kadın, ya da her bir deney için farklı bir baraj kullanımına tekrarlanan tedaviler atlar. Bu protokol ayrıca hassas çöp eşleştirilmiş ve aynı yaştaki yenidoğanların çalışma sağlar. Temsili veriler bu protokolün bir başka önemli gücü, AIH, teslim olarak, nöral kök hücre biyolojisinde güçlü ve tutarlı bir biyolojik tepkiyi ortaya çıkaran hangi ile yani Çabukluğu göstermektedir. Bu doku ve hücre düzeyinde biyolojik ka ortaya çıkarmak için bu protokol için bir emsal oluştururHücre biyolojisi değiştiren cal değişir. Bu rapor AIH için kemirgen yavrular yanı sıra neurospheres olarak yetiştirilen SVZ hücrelerin nüfus analizi sergilemek için kullanılan ayrıntılı prosedürler açıklayacağım.

<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.

Tarihsel verilere dayalı ilk deneyler, 1 dk döngüsü uzunlukları kullanılarak yapılmıştır. Yukarıdaki 2. Adımda gerçekleştirilen sonraki kalibrasyon dayanarak, bunun% 21 başlangıca dönmek için benzer bir zaman aldı, odasında O 2 seviyesi 1 dk sonrası hipoksi flushing&#39;in 13% olduğu belirlenmiştir edildi ve. Bununla birlikte, 2 dakika döngüsü 10% elde oksijen ve &quot;taban&quot; döngüsü sırasında% 21 bir dönüş hem de yeterli olmuştur. Daha sonra, 2 dakika döngü uzunlukla...

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

Bu makale doğum sonrası gün 1-8 fare veya sıçan yavrular aralıklı hipoksi kısa süreli uygulanması için yöntem anlatılmaktadır. Bu yaklaşım etkili bir hipoksi maruz kalma 30 dakika içinde hasat edilir kültürlü nöral progenitör hücreler üzerindeki sağlam doku düzeyi &quot;etkisi priming&quot; ortaya çıkarır.

Teslimatı<em&gt; İn Vivo</em&gt; Akut Aralıklı hipoksi Yenidoğan Kemirgenler Başbakan subventricular bölgesi kökenli Sinir Progenitör Hücre Kültürleri için

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 Görüntüleme.  University of Florida.  Bu makale doğum sonrası gün 1-8 fare veya sıçan yavrular aralıklı hipoksi kısa süreli uygulanması için yöntem anlatılmaktadır. Bu yaklaşım etkili bir hipoksi maruz kalma 30 dakika içinde hasat edilir kültürlü nöral progenitör hücreler üzerindeki sağlam doku düzeyi "etkisi priming" ortaya çıkarır. 

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.

In vivo Nöromimarlık çalışmaları için tasarlanan nöronal öncülerin intraventriküler nakli

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

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.

Perinatal İskemi Hipoksik İskemik Ensefalopati Modeli

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

Biyoloji

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

Yenidoğan subventricular Bölge Elektroporasyon

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

Nörobilim

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.

Fare Akut Beyin Dilimleri Neuroblast Göç vivo Doğum Sonrası Elektroporasyonla ve Time-lapse Görüntüleme

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.

Prematüre Modelleme Ensefalopati Sıçanlarda İçi amniyotik Lipopolisakkarid ile Prenatal Hipoksi-iskemi kullanma

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

Tıp

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.

Dönem Yenidoğan Sıçanlarda Kök Hücrelerin İntratrakeal Aşılanması

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.

Yenidoğan Farelerde intravenöz enjeksiyonlar

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.

Yenidoğan Kemirgenlerinde Akciğer Hastalığını Tedavi Etmek İçin İlaçların İntratrakeal İnstilasyonu İçin Minimal İnvaziv Bir Yöntem

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 ...

Nöral Kök ve Oligodendrosit Progenitör Hücre İzolasyonu için Sıçan "Beyin Sağım"

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.

Yazar Spotlight: Yeni Bir Beyin Sağım Protokolü Kullanarak Canlı Hayvanlardan Nöral Kök ve Progenitör Hücrelerin Toplanması 

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.

Canlı Sıçanlardan Nöral Kök Hücrelerin ve Oligodendrosit Progenitör Hücrelerin İzolasyonuna Yönelik "Beyin Sağım" Yöntemi

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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Bu videodaki bölümler

In vivo Nöromimarlık çalışmaları için tasarlanan nöronal öncülerin intraventriküler nakli

Perinatal İskemi Hipoksik İskemik Ensefalopati Modeli

Yenidoğan subventricular Bölge Elektroporasyon

Fare Akut Beyin Dilimleri Neuroblast Göç vivo Doğum Sonrası Elektroporasyonla ve Time-lapse Görüntüleme

Prematüre Modelleme Ensefalopati Sıçanlarda İçi amniyotik Lipopolisakkarid ile Prenatal Hipoksi-iskemi kullanma

Dönem Yenidoğan Sıçanlarda Kök Hücrelerin İntratrakeal Aşılanması

Yenidoğan Farelerde intravenöz enjeksiyonlar

Yenidoğan Kemirgenlerinde Akciğer Hastalığını Tedavi Etmek İçin İlaçların İntratrakeal İnstilasyonu İçin Minimal İnvaziv Bir Yöntem

Canlı Sıçanlardan Nöral Kök Hücrelerin ve Oligodendrosit Progenitör Hücrelerin İzolasyonuna Yönelik "Beyin Sağım" Yöntemi

Canlı Sıçanlardan Nöral Kök Hücrelerin ve Oligodendrosit Progenitör Hücrelerin İzolasyonuna Yönelik "Beyin Sağım" Yöntemi