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

참고 :이 프로토콜의 모든 동물 절차는 플로리다 기관 동물 관리 및 사용위원회 (IACUC)의 대학의 승인을 실시하고 &#39;실험 동물의 관리 및 사용을위한 안내서&#39;을 준수하고 있습니다. 1. 기본 실험 세트까지 간헐적 저산소증 관리하기 전에 <ol><li> 다른 실험 목적을위한 5,13,14 성인 설치류만큼 동일하게 전신 마우스 plethysmograph 챔버를 사용하여 상이한 가스 혼합물에 새끼 12을 노출. 챔버는 3-4 마우스 새끼 또는 1-2 래트 새끼를 수용하기 충분한 크기 인 4 인치 직경 (부피 = 450 ml)에,이. 여기에, AIH 프로토콜은 출생 후 하루 1-8 (P1 ~ P8) 세 신생아에게 전달됩니다. </li><li> 21 %에서 챔버 산소를 유지 21 % &quot;기준&quot;가스를 제공하기 위해 압축 공기 가스 탱크를 사용합니다. &quot;상대적 저산소증&quot;을 달성하기 위해, 10 % 산소 / 90 %의 질소 가스 탱크를 사용한다. 하지E : 플라스틱 배관 (3/16 &quot;직경)는 유량계 가스 탱크를 연결한다. 따라서, 유량계에 대한 입력은 하나의 튜브 및 저산소증 기준선 기류 각 이루어져있다. </li><li> 다음과 같이 가스 배관을 구성합니다. <ol><li> 각 챔버에 1 L / 분의 흐름을 가능하게하는 바이어스 유동 조절기로 유량계에서 플라스틱 배관 (1/8 &quot;직경)을 실행 (즉, 4 L / 분 (4) 챔버가 사용되는 경우). </li><li> 혈량 측정법 챔버에 바이어스 유량 조절기에서 플라스틱 튜브 (1/8 &quot;직경)를 실행합니다. 기류는 1에서 L / 분으로 각각의 챔버는 가스 교환이 비 생리적 스트레스 유발 및 행동의 관측에 기초하여 동물에 것으로 간주되는 소정 유량이다. </li></ol></li><li> 어떠한 가스 유동이 프로토콜 동안 사용되지 않을, 또는 혈량 측정법 챔버 중 다른 챔버로 빠져 나가지 않도록 캡 챔버 및 상기 바이어스 부에서 유동하지 않는 모든 연결 및 모든 미사용 개구부를 밀봉. </li><li> (실을 배치의) 37 ° C로 평형화 또는 체온을 허용하도록 챔버에 동물을 성공시키는 종래 인큐베이터. </li></ol> 간헐적 저산소증에 대한주기 시간 2. 교정 <ol><li> 공기 탱크 및 유량계 사이, 모든 배관을 검사 적절한 연결 m 바이어스 흐름 부, 그리고 바이어스 흐름 부와 혈량 측정법 챔버 (들)를 흐른다. 예를 들어, 사용하지 않는 미사용 바이어스 유동 부의 아웃 라인 및 챔버 개구를 체크 : 모든 설명 선과 클로저 단계적 수표 접속 확인 단계 1에서 설명한 바와 같이 접속이 설정된다. </li><li> 휴대용 O 2m로 주변 O2 센서를 연결 한 후 혈량 측정법 챔버 뚜껑에 센서를 부착합니다. </li><li> 모두 가스 탱크를 열고 가스의 오프 사이클 흐름을 고정하는 플라스틱 튜브로 절연 수술, 긴 처리 지혈 한 쌍을 연결합니다. 이러한 방식으로, 한 번에 하나의 가스 탱크 챔버 당 1 L / min의 가스 흐름을 제공한다. &quot;저산소증&quot;가스 믹스가 반대로 챔버로 전달되고, 반면 &quot;기준선&quot;가스 믹스 클램프. </li><li> 10 %의 목표 레벨에 도달하고 21 %로 돌아 챔버 O 2에 필요한 시간을 기록한다. 다음 프로토콜 전달을위한 기록 된 시간을 활용합니다. </li></ol> 급성 간헐적 저산소증 3. 관리 <ol><li> 2.1 단계에 설명 된대로 모든 배관 및 연결을 검사합니다. </li><li> 혈량 측정법 챔버 (들)에서 신생아를 놓고 챔버베이스로 혈량 측정법 챔버 뚜껑을 수용. 사용하지 않는 실 연결의 적절한 O- 링 씰 씰입니다뿐만 아니라 폐쇄를 확인합니다. 이 단계는 가스 혼합물의 적절한 공급을 보장하는 데 중요합니다. </li><li> 새끼를 보유 챔버가 37 ° C 배양기로 처리 할 수​​ 놓고 문을 닫습니다. 챔버는 이전 프로토콜을 시작으로 37 ° C의 평형을 허용합니다. incubat 37 ° C에서 제어 동물을 유지또는 실내 공기의 산소에서. </li><li> 모두 가스 탱크를 열고 가스의 오프 사이클 흐름을 고정하는 플라스틱 튜브로 절연 수술, 긴 처리 지혈 한 쌍을 사용합니다. 이러한 방식으로, 한 번에 하나의 가스 탱크 챔버 당 1 L / min의 가스 흐름을 제공한다. </li><li> 정밀 시간주기에 휴대용 타이머를 사용함으로써 챔버로의 가스 유동의 교호 결과, 튜브 사이에 지혈을 전환하는 시간을 표시. </li><li> 이러한 2 단계, 20 사이클 프로토콜의 모든 단계를 표시하기 위해도 1에 제공된 것과 같은 처리 로그를 이용하여 각주기의 완료를 기록한다. </li><li> 동물이 지정된 범위 (37 ° C 결과 인큐베이터에있는 설정입니다 33 ~ 35 ° C)에서의 유지를 보장하기 위해 모든 사이클 변화에 인큐베이터 온도를 모니터링합니다. </li><li> 밀접하게 동물은 발성의 변경된 수준으로 볼 수 고통없이 사이클을 견딜 수 있도록 프로토콜을 통해 강아지의 활동을 모니터링사지 활동이나 압연. </li></ol> 뇌실 영역에서 줄기 / 전구 세포의 4. 분리 및 문화 주 : 대조군과 비교 AIH 다음 neurosphere 포메이션에 변화는 조직 - 및 세포 수준의 변화를 유발하는이 프로토콜의 효능을 입증 엔드의 일례이다. <ol><li> 세포를 형성 neurosphere를 격리하기 위해, AIH가 완료된 직후 챔버에서 마우스 또는 쥐 새끼를 제거합니다. 프로토콜 종료 30 분 이내에 IACUC 프로토콜에 따라 동물을 희생. </li><li> 이전 방법 장 (15, 16)과 별도의 비디오 프로토콜 (8)에 발표 된 바와 같이, SVZ 조직을 수확 neurosphere 문화에 배치하고 표준 통로 파생 neurosphere를의 확장을 실시하고 있습니다. </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...

이 방법의 목적은 재현성과 효과적으로 신생아 설치류에 전신 낮아 주변 산소의 간헐적 인 관찰을 제공하는 것입니다. 줄기 세포 생물학 조작 간헐적 저산소증 (IH)를 사용하는 이론적 근거는 성장 배지 2 O의 함유량이 변경되는 시험 관내 세포 배양 실험에서 유래. 20 %의 &quot;표준&quot;조건 O 2, 증가 확산의 3 % O 2 결과 줄기 / 전구 세포 집단 세포의 확장 된 문화에 비해 특히, 세포 사멸을 감소 신경 수익률 1,2 증가했다. 이 그룹은 전신 IH의 관리에 상당한 경험을 가지고 있으며, 호흡 소성 3-7 IH의 역할에 대한 광범위한 연구를 수행하고있다. 이 작품, 만성 IH은 설치류 CNS 8-10에 신경을 증가하는 최근의 연구 결과는 생체 내에서 급성의 탐사를위한 기초를 형성전처리 자극으로 저산소증 (예., 조직 수확 이전에) 신경 줄기 / 전구 세포 (NPC들) (11)의 연속 문화. 마우스 새끼가 급성 간헐적 저산소증 (AIH)의 개요 (&lt;1 시간) 기간에 노출되었을 때 놀랍게도, 뇌실 영역 (SVZ)에서 수확 된 세포는 크게 neurosphere를 또는 부착 단층 세포와 같은 확장을위한 용량을 증가했다. AIH 프로토콜은 또한 &quot;신경 운명&quot;전사 인자 (PAX6)의 발현 증가와 관련이 있었다. 따라서, 생체 내에서 AIH 프로토콜이 &quot;소수&quot;의 NPC 전에 문화 할 수있는 수단을 제공 할 수있다. 예를 들어,이 방법에 대한 응용 프로그램은 손상된 중추 신경계에 이전에 이식 세포 집단을 확장하거나 이전의 시험 관내 실험에 배양 된 세포의 신경 세포 분화를 증가 포함 할 수있다. 또한,이 전신 전달을하기 때문에, 어떠한기관, 조직 또는 세포는 유사한 연구를위한 후보입니다. 따라서 작성된 프로토콜은 작은 포유류 간헐적 산소 조작에 관한 연구의 다양한 잠재적으로 적용될 수있다. 이 방법의 특정 이점이있다. 게시 된 다른 연구에서 신생아가 치료에 앞서, 만성 투여 덜 처리를 할 수 있습니다 기압 성 챔버의 댐과 쓰레기로 취급하고, 치료 9시 산모의 접촉을 유지 하였다. 현재의 접근법은 사육 여성, 또는 각각의 실험에 대해 다른 댐의 사용을 반복 치료를 무시. 이 프로토콜은 또한 정확한 쓰레기 일치 및 연령대 신생아의 연구를 할 수 있습니다. 대표적인 데이터는이 프로토콜의 또 다른 키 강도, AIH는 전달과 같은 신경 줄기 세포 생물학에서 강력하고 일관된 생물학적 반응을 이끌어있는 즉 신속성을 보여줍니다. 이 조직 - 및 세포 수준 biologi을 유도하기 위해이 프로토콜에 대한 선례를 확립세포 생물학을 변경 CAL 변경됩니다. 이 보고서에 AIH 설치류뿐만 아니라 새끼 neurosphere를 성장 중 SVZ 세포의 집단 분석을 노광에 사용되는 세부 과정을 개설 할 것이다.

<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.

기록 데이터에 기초하여 초기의 실험은, 1 분의 사이클 길이를 이용하여 수행 하였다. 상기 단계 2에서 수행되는 후속 캘리브레이션에 기초하여, 그것이 21 % 기준선으로 돌아 유사한 시간 걸렸다, 챔버 내의 O 2 레벨이 1 분 후 저산소증 플러싱에서 13 %라고 결정 하였다. 그러나, 2 분주기의 10 %를 달성 산소 및 &quot;기준&quot;사이클 동안 21 %로 복귀시키기에 충분한 양이었다. 이어서, 2 분주기...

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

이 문서에서는 출생 후 하루 1-8 마우스 또는 쥐 새끼에 간헐적 저산소증의 짧은 기간을 관리하는 방법을 설명합니다. 이러한 접근은 효과적 저산소증 노출 30 분 이내에 수확 배양 신경 전구 세포에 대한 강력한 조직 수준 &quot;프라이밍 효과&quot;를 도출한다.

배달<em&gt; 생체</em&gt; 급성 간헐적 저산소증 신생아 설치류에서 국무 뇌실 지역 유래 신경 전구 세포 배양에

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

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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 조회수.  University of Florida.  이 문서에서는 출생 후 하루 1-8 마우스 또는 쥐 새끼에 간헐적 저산소증의 짧은 기간을 관리하는 방법을 설명합니다. 이러한 접근은 효과적 저산소증 노출 30 분 이내에 수확 배양 신경 전구 세포에 대한 강력한 조직 수준 "프라이밍 효과"를 도출한다. 

비디오: 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.

생체 내 신경 아키텍처 연구를 위한 엔지니어링된 신경 전구체의 내 측 통 이식

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

연구

유전학

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 국소 빈혈의 Hypoxic 허혈성 뇌증 모델

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

생물학

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

신생아 Subventricular 지대 Electroporation

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

신경과학

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.

마우스 급성 뇌 조각의 Neuroblast 마이그레이션의 생체 내 출생 일렉트로 및 시간 경과 영상

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.

미숙아의 모델링 뇌증은 흰쥐의 내부 - 양수 리포 폴리 사카 라이드와 태아 저산소증 - 허혈을 사용하여

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

의학

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.

신생아 쥐 기간에 줄기 세포의 내 치 통 주입

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.

신생아 마우스의 정맥 주사

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.

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 ...

신경 줄기 및 희소돌기아교세포 전구 세포 분리를 위한 쥐 "뇌 착유"

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.

저자 스포트라이트: Collecting Neural Stem and Progenitor Cells from Live Animals Using a Novel Brain Milking Protocol (새로운 뇌 착유 프로토콜을 사용하여 살아있는 동물에서 신경 줄기 및 전구 세포 수집) 

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.

살아있는 쥐에서 신경 줄기 세포와 희소돌기아교세포 전구 세포를 분리하기 위한 "뇌 착유" 방법

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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