저자는 금융 지원을위한 SMA의 NINDS, FightSMA, 가족을 인정하고 싶습니다. SEGL은 NINDS 훈련 보조금 # 5T32NS077984-02에 의해 지원됩니다.

프로토콜에 나열된 모든 절차는 오하이오 주립 대학의 동물 사용 및 관리위원회 (IACUC) 연구소 승인되었습니다. 작업 1. 준비 <ol><li> 깔짚, 해부 현미경, 90 °로 주사 (광원의 사용 각도로 위치 될 수 광원으로부터 댐을 분리하는, 빈 케이지 마우스 새끼 마취 젖은 얼음 수집 &#160; 주사 부위에 각도는 정맥), 주사에 대한 동물을 배치 깨끗한 표면, 면봉, 3/8 &quot;30 G 바늘 (동물 당), 1 % 에반스 블루 염료 3/10 CC 인슐린 주사기 (가린다 교육을위한 인산염 완충 생리 식염수 (PBS)) 솔루션을했다. </li><li> 새끼를 조작하면서 별도의 케이지에 댐을 제거합니다. </li></ol> 2. 사출 절차 <ol><li> 동물을 마취하는 30 ~ 60 초 동안 얼음물에 직접 하나의 강아지를 놓습니다. t 방치하지 마십시오인해 심실 세동, 조직의 저산소증과 대사성 산증을 포함하여 저체온증과 관련된 합병증의 위험에 너무 오래 얼음에 그는 동물. 참고 : 우리의 경험은 30 ~ 60 초 분사를 허용하는 강아지의 움직임을 느리게하기에 충분하다는 것이다. 깊은 마취가 요구되는 경우, 예컨대 1 ~ 2 %의 이소 플루 란과 같은 흡입제가 적절할 수있다. </li><li> 동물이 얼음에 있지만, 에반스 블루 염료의 30 μL와 주사기를로드합니다. </li><li> 동물이 완전히 마취되면, 여전히 호흡하는 동안 얼음에 운동 부족에 의해 확인 현미경으로 이동합니다. 오른손 주입 들어, 오른쪽으로 동물의 총구에 직면한다. 이어 버드는 색인과 중간 손가락 (그림 1) 사이가되도록 이어 버드에 총구와 왼쪽 가운데 손가락 꼬리에 왼쪽 검지 손가락을 놓습니다. </li><li> 단지 피부가 조작 될 때 이동하는 표면 모세 혈관의 이어 버드로 전방 검사합니다. 이 모세관하지만, 대상이 아니다식별은 시간 정맥 식별을 위해 중요하다. 다음에 관계없이 피부의 위치에 고정되어 모세관 열등 어두운 그림자가 정맥을 찾습니다. 시간 정맥, 그림자가 나타납니다 지느러미에 복부 실행하고, 경정맥 (그림 1)에 공급한다. </li><li> 바늘 베벨 위로 시간적 정맥을 입력합니다. 올바르게 삽입하면 바늘 베벨 피부를 통해 혈액으로 채워 볼 수있다. 그런 다음 천천히 플런저를 우울하게하고 노트는 얼굴의 측면 아래 정맥의 창백. </li><li> 바늘이 주입 물 역류를 방지하기 위해 첨가 10-15 초 동안 정맥 내 유지하도록 허용. </li></ol> 3. 주입 후 <ol><li> 적절한 주입 한 후, 강아지는 거의 즉시 파란색 설정해야합니다. 바늘을 제거하고 혈액 응고 될 때까지 주사 부위에 면봉을 적용하는 부드러운 힘을 사용합니다. </li><li> 고통의 흔적이 강아지를 모니터링합니다. 강아지 2 ~ 3 분 복구하고 rewarm 허용, R강아지가 케이지에 반환하기 전에, 의식 수직 및 이동 때 ecognize. 컵 탐정의 장갑을 낀 손에 새끼 필요한 경우 복구에 도움이되는 적절한 온기를 제공합니다. 선택적으로, 주입 된 새끼 온난화 촉진하는 홈 케이지 하에서 가열 패드를 배치. 일반적으로 에반스 블루 염료를 주입 한 후, 새끼를 안락사. 시험 물질을 주입 할 때 염료를 포함하지 마십시오. </li><li> 다시 홈 케이지에 강아지를 놓고 강아지가 댐에 의해 reacceptance을 보장하기 위해 침구 및 / 또는 nestlet로 코팅되어 있는지 확인합니다. </li><li> 불임을 유지하기 위해 각각의 강아지에 대한 새 주사기와 면봉을 사용합니다. </li></ol>

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저자가 공개하는 게 없다.

CNS 또는 몸 전체 에이전트의 혈관 배달 질환의 신생아 쥐 모델에서 어렵다. 설명 프로토콜은 정맥 최소한의 장비 요구 사항에 신생아 쥐에 솔루션을 관리 할 수​​있는 빠르고, 비교적 비 침습적 방법입니다. 시간적 얼굴 정맥을 육안으로 볼 수 있지만, 주사는 특히 unpracticed 인젝터, 현미경 및 광섬유 광원의 사용과 높은 정확도를 가질 수있다. 신생아 마우스에서 혈관 내 주사는 높은 성공률 <sup...

소아 질병의 쥐 모델에서 중추 신경계 (CNS)에 대한 치료제의 배달는 도전 할 부분이 남아있다. 모델 신생아 질병 상태를 직접 CNS 내에서 적절한 구조에 삽입하기 어려울 수 있으므로 소형 및 발달 미숙 한 것을 마우스. 치료제의 혈관 내 주입 CNS 1-5과 3,5-9 망막을 포함한 몸 전체에 세포, 약물, 또는 바이러스 벡터를 제공 할 수있는 비 침습적, 내약성 방법이다. 이전 출판물은 해부 현미경 11, 12 않고, transilluminator가 10, 11을 사용하여 시간적 얼굴 정맥 주입을 설명, 또는 두 사람을 요구하는 것은 (10)를 삽입 할 수 있습니다. 한 개인이 새끼를 주입 할 수 있으며, 광원은 시간적 정맥이 강아지를 만지고 고정 표면 외과 테이프의 필요성 또는 강아지의 부착을 제거하지 않은 볼 때문에 프로토콜에서 설명 주입 기술은 유리한transilluminator가 11 등. 마우스에 아데노 - 관련 바이러스 벡터 혈청 형 9 (AAV9)의 전달은 뇌와 척수 (그림 1)를 통해 신경 세포와 성상 세포에 강력한 표현을 생성합니다. 피상적 시간적 안면 정맥 내로 바이러스 벡터의 전달을 확실하게 혈관 신경 근육 질환 소아 척수 근육 위축 (SMA) 2,4,13,14을 치료하는 신생아 마우스에서 다양한 연구에 사용 된 궁극적으로 처리 된 마우스의 수명을 증가되었다. 신생아 쥐의 혈관 내 주입도 효과적으로 말초 신경계 및 말초 기관 (그림 2)을 대상으로합니다. AAV의 주입 후, 후근 신경절, 간, 심장, 골격근, 폐, 그리고 창자의 myenteric 신경 얼기의 전달은 1,3,6,7,15을 관찰되었다. CNS와 주변의 대폭적인 전달은 글로벌 표현을 필요로하는 질병에 대한 분사 이상이 방법을 만든다이러한 고셔병 (16) 및 기타 리소좀 저장 질환 17, 18, ​​판자의 질병과 관련된 신경 ceroid lipofuscinoses, 19 Bardet-Biedl 증후군, 유아 (20)에서 발생하는 증상의 발병에 유전 적 다중 시스템 장애로 형질 전환 유전자,. 혈관 신생 생쥐에 주입은 모델링 시스템 전체 소아 질환의 신규 한 방법으로 고려되어야한다. 이 기술은 큰 동물 모델 5,21로 번역 한 내용 및 혈관 내 주입 이미 치료제를 전달 임상 적으로 허용되는 방법으로 존재한다. 현재의 프로토콜은 출생 후 하루 2 사출 늦어도 피상적 인 시간적 얼굴 정맥을 통해 신생아 쥐에 에이전트를 도출하는 간단하고 효율적인 방법을 설명하는 하나의 완성 할 수있다, 개인 연습을 잘 새끼와 어미 모두 허용됩니다. 새끼 최소한의 고통을 경험하고 신속하게 복구 할 수 있습니다. ImportanTLY, 성공적인 주입은 관리 에이전트의 글로벌 배달 될 것입니다. 이 프로토콜은 바이러스 벡터, 신생아 쥐에 제약 제제 또는 세포의 전달에 적합하다.

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intravenous injections in neonatal mice

정맥 주사는 치료제를 제공하기 위해 임상 적으로 적용 가능한 방법이다. 성인 설치류 큰 동물의 경우, 정맥 주사는 기술적으로 가능하고 일상이다. 그러나 일부 마우스 모델은 잠재적 인 치료의 관리가 어려워 빠른 진행과 질병의 조기 발병을 가질 수 있습니다. 시간 (또는 안면) 정맥은 마우스에서 이어 버드에 전방 및 해부 현미경을 사용하여 머리의 어느 한쪽에 생후 명확 처음 이틀 동안 표시된다. 이 윈도우 동안, 시간적 정맥 50 μl의 최대 양으로 주입 될 수있다. 주사는 안전하고 잘 새끼와 어미 모두 허용합니다. 전형적인 주입 과정은 강아지가 홈 케이지로 복귀 된 후 1-2 분 이내에 완료된다. 제 생후함으로써 정맥 시각화 어렵고 주입 과정은 기술적으로 불안정해진다. 이 기술은 아데노 - 관련 바이러스 (AAV) VECT의 전달을 위해 사용되어왔다차례로 선택한 바이러스 혈청 형에 따라 동물의 수명을 거의 몸 전체, 안정적인 형질 전환 유전자 발현을 제공 할 수 있습니다 ORS.

적절한 주입하는 동안, 정맥은 순간적으로 분명, 또는 희게을 설정해야합니다. 염료 주입하면 전체 강아지 초 ​​이내에 파란색 설정해야합니다. 부적절한 분사가 발생한 경우, 종종 주사 부위 밖으로 누출 될 머리 또는 목 주입 물에서 농축 피하 볼 러스있다. 부적절한 주사는 또한 목 주위에 멍이의 모양이 발생할 수 있습니다. (분사가 완전히 정맥에 배달되지 않은 IE) 일반적으로 더 아?...

Watch this Scientific Journal Video about Intravenous Injections in Neonatal Mice at JoVE.com

Intravenous Injections in Neonatal Mice

소아 질환의 동물 모델은 초기 발병과 적극적인 질병의 진행을 경험할 수 있습니다. 임상 어린 마우스 모델에 관련된 치료 전달 기술적 어려울 수있다. 이 프로토콜은 생후 두 생후 일 이내 신생 쥐 비 침습적 정맥 주입 방법을 설명한다.

신생아 마우스의 정맥 주사

Intravenous injection is a clinically applicable manner to deliver therapeutics. For adult rodents and larger animals, intravenous injections are ...

Research

JoVE Journal

Biology

57.5K 조회수.  Ohio State University.  소아 질환의 동물 모델은 초기 발병과 적극적인 질병의 진행을 경험할 수 있습니다. 임상 어린 마우스 모델에 관련된 치료 전달 기술적 어려울 수있다. 이 프로토콜은 생후 두 생후 일 이내 신생 쥐 비 침습적 정맥 주입 방법을 설명한다. 

비디오: Intravenous Injections in Neonatal Mice

Heterotopic Heart Transplantation in Mice

The mouse heterotopic heart transplantation has been used widely since it was introduced by Drs. Corry and Russell in 1973. It is particularly valuable for studying rejection and immune response now that newer transgenic and gene knockout mice are available, and a large number of immunologic reagents have been developed. The heart transplant model is less stringent than the skin transplant models, although technically more challenging. We have developed a modified technique and have completed over 1000 successful cases of heterotopic heart transplantation in mice. When making anastomosis of the ascending aorta and abdominal aorta, two stay sutures are placed at the proximal and distal apexes of recipient abdominal aorta with the donor s ascending aorta, then using 11-0 suture for anastomosis on both side of aorta with continuing sutures. The stay sutures make the anastomosis easier and 11-0 is an ideal suture size to avoid bleeding and thrombosis.
When making anastomosis of pulmonary artery and inferior vena cava, two stay sutures are made at the proximal apex and distal apex of the recipient s inferior vena cava with the donor s pulmonary artery. The left wall of the inferior vena cava and donor s pulmonary artery is closed with continuing sutures in the inside of the inferior vena cava after, one knot with the proximal apex stay suture the right wall of the inferior vena cava and the donor s pulmonary artery are closed with continuing sutures outside the inferior vena cave with 10-0 sutures. This method is easier to perform because anastomosis is made just on the one side of the inferior vena cava and 10-0 sutures is the right size to avoid bleeding and thrombosis. In this article, we provide details of the technique to supplement the video.

The mouse heterotopic heart transplantation model has been proven by many investigators to be an important method for studying mechanisms of rejection and immune response. However, the techniques involved are still challenging. By modifying standard techniques we have had success with more than 1000 transplants.

마우스의 Heterotopic 심장 이식

The mouse heterotopic heart transplantation has been used widely since it was introduced by Drs. Corry and Russell in 1973. It is particularly valuable ...

연구

생물학

Small Bowel Transplantation In Mice

Since 1990, the development of tacrolimus-based immunosuppression and improved surgical techniques, the increased array of potent immunosuppressive medications, infection prophylaxis, and suitable patient selection helped improve actuarial graft and patient survival rates for all types of intestine transplantation. Patients with irreversible intestinal failure and complications of parenteral nutrition should now be routinely considered for small intestine transplantation. However, Survival rates for small intestinal transplantation have been slow to improve compares increasingly favorably with renal, liver, heart and lung. The small bowel transplantation is still unsatisfactory compared with other organs. Further progress may depend on better understanding of immunology and physiology of the graft and can be greatly facilitated by animal models. A wider use of mouse small bowel transplantation model is needed in the study of immunology and physiology of the transplantation gut as well as efficient methods in diagnosing early rejection. However, this model is limited to use because the techniques involved is an extremely technically challenging. We have developed a modified technique. When making anastomosis of portal vein and inferior vena cava, two stay sutures are made at the proximal apex and distal apex of the recipient s inferior vena cava with the donor s portal vein. The left wall of the inferior vena cava and donor s portal vein is closed with continuing sutures in the inside of the inferior vena cava after, after one knot with the proximal apex stay suture the right wall of the inferior vena cava and the donor s portal vein are closed with continuing sutures outside the inferior vena cave with 10-0 sutures. This method is easier to perform because anastomosis is made just on the one side of the inferior vena cava and 10-0 sutures is the right size to avoid bleeding and thrombosis.  In this article, we provide details of the technique to supplement the video.

The mouse small bowel transplantation model has been recognized as an important tool to study mechanismes of immune rejection and screen new immunosuppressive drugs. However, this model is limited to use because the techniques involved is an extremely technically challenge. Now we introduce the modified technique.

마우스에 작은 창자 이식

Since 1990, the development of tacrolimus-based immunosuppression and improved surgical techniques, the increased array of potent immunosuppressive ...

Transverse Aortic Constriction in Mice

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model for pressure overload-induced cardiac hypertrophy and heart failure.1 TAC initially leads to compensated hypertrophy of the heart, which often is associated with a temporary enhancement of cardiac contractility. Over time, however, the response to the chronic hemodynamic overload becomes maladaptive, resulting in cardiac dilatation and heart failure.2 The murine TAC model was first validated by Rockman et al.1, and has since been extensively used as a valuable tool to mimic human cardiovascular diseases and elucidate fundamental signaling processes involved in the cardiac hypertrophic response and heart failure development. When compared to other experimental models of heart failure, such as complete occlusion of the left anterior descending (LAD) coronary artery, TAC provides a more reproducible model of cardiac hypertrophy and a more gradual time course in the development of heart failure. Here, we describe a step-by-step procedure to perform surgical TAC in mice. To determine the level of pressure overload produced by the aortic ligation, a high frequency Doppler probe is used to measure the ratio between blood flow velocities in the right and left carotid arteries.3, 4 With surgical survival rates of 80-90%, transverse aortic banding is an effective technique of inducing left ventricular hypertrophy and heart failure in mice.

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model to study mechanisms underlying cardiac hypertrophy and heart failure development. Here, we describe procedures to constrict the aorta to create a reproducible degree of cardiac hypertrophy in mice.

마우스의 가로 대동맥 수축

Transverse aortic constriction (TAC) in the mouse is a commonly used experimental model for pressure overload-induced cardiac hypertrophy and heart ...

Ambulatory ECG Recording in Mice

Telemetric ECG recording in mice is essential to understanding the mechanisms behind arrhythmias, conduction disorders, and sudden cardiac death. Although the surface ECG is utilized for short-term measurements of waveform intervals, it is not practical for long-term studies of heart rate variability or the capture of rare episodes of arrhythmias. Implantable ECG telemeters offer the advantages of simple surgical implantation, long-term recording of electrograms in ambulatory mice, and scalability with simultaneous recordings of multiple animals. Here, we present a step-by-step guide to the implantation of telemeters for ambulatory ECG recording in mice. Careful adherence to aseptic technique is required for favorable survival results with the possibility of implantation and recording from weeks to months. Thus, implantable ECG telemetry is a valuable tool for detection of critical information on cardiac electrophysiology in ambulatory animal models such as the mouse. 

Telemetric ECG has emerged as an essential tool in evaluating animal models for cardiac arrhythmias and sudden cardiac death. Here, we present a stepwise guide to telemetric ECG recordings for application in long-term ambulatory ECG monitoring in mice.

마우스의 외래 ECG 기록

Telemetric ECG recording in mice is essential to understanding the mechanisms behind arrhythmias, conduction disorders, and sudden cardiac death. Although ...

Intravenous Microinjections of Zebrafish Larvae to Study Acute Kidney Injury

In this video article we describe a zebrafish model of AKI using gentamicin as the nephrotoxicant.  The technique consists of intravenous microinjections on 2 dpf zebrafish. This technique represents an efficient and rapid method to deliver soluble substances into the bloodstream of zebrafish larvae, allowing for the injection of 15-20 fish per hour. In addition to AKI studies, this microinjection technique can also be used for other types of experimental studies such as angiography.  We provide a detailed protocol of the technique from equipment required to visual measures of decreased kidney function. In addition, we also demonstrate the process of fixation, whole mount immunohistochemistry with a kidney tubule marker, plastic embedding and sectioning of the larval zebrafish. We demonstrate that zebrafish larvae injected with gentamicin show morphological features consistent with AKI: edema, loss of cell polarity in proximal tubular epithelial cells, and morphological disruption of the tubule.

We describe a technique of microinjecting the aminoglycoside, gentamicin, into 2 days post-fetilization (dpf) zebrafish larvae to induce acute kidney injury (AKI). We also describe a method for whole mount immunohistochemistry, plastic embedding and sectioning of zebrafish larvae to visualize the AKI mediated damage.

급성 신장 상해를 공부하기 위해 Zebrafish 애벌레의 정맥 Microinjections

In this video article we describe a zebrafish model of AKI using gentamicin as the nephrotoxicant.  The technique consists of intravenous microinjections ...

Isolation and Culture of Pulmonary Endothelial Cells from Neonatal Mice

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells (HUVECs) have shown to be convenient, easy to obtain and culture, and thus are the most widely studied endothelial cells. However, for research focused on processes like angiogenesis, permeability or many others, microvascular endothelial cells (ECs) are a much more physiologically relevant model to study 2. Furthermore, ECs isolated from knockout mice provide a useful tool for analysis of protein function ex vivo. Several approaches to isolate and culture microvascular ECs of different origin have been reported to date 3-7, but consistent isolation and culture of pure ECs is still a major technical problem in many laboratories. Here, we provide a step-by-step protocol on a reliable and relatively simple method of isolating and culturing mouse lung endothelial cells (MLECs). In this approach, lung tissue obtained from 6- to 8-day old pups is first cut into pieces, digested with collagenase/dispase (C/D) solution and dispersed mechanically into single-cell suspension. MLECS are purified from cell suspension using positive selection with anti-PECAM-1 antibody conjugated to Dynabeads using a Magnetic Particle Concentrator (MPC). Such purified cells are cultured on gelatin-coated tissue culture (TC) dishes until they become confluent. At that point, cells are further purified using Dynabeads coupled to anti-ICAM-2 antibody. MLECs obtained with this protocol exhibit a cobblestone phenotype, as visualized by phase-contrast light microscopy, and their endothelial phenotype has been confirmed using FACS analysis with anti-VE-cadherin 8 and anti-VEGFR2 9 antibodies and immunofluorescent staining of VE-cadherin. In our hands, this two-step isolation procedure consistently and reliably yields a pure population of MLECs, which can be further cultured. This method will enable researchers to take advantage of the growing number of knockout and transgenic mice to directly correlate in vivo studies with results of in vitro experiments performed on isolated MLECs and thus help to reveal molecular mechanisms of vascular phenotypes observed in vivo.

Here, we describe a protocol for isolation and culture of murine pulmonary endothelial cells. This method comprises mechanic and enzymatic lung tissue dissociation as well as a 2-step purification process using anti-PECAM-1 and anti-ICAM-2 antibodies conjugated to magnetic beads, which produces a pure endothelial cell population of mostly microvascular origin.

신생아 마우스의 폐 내피 세포의 분리 및 문화

Endothelial cells provide a useful research model in many areas of vascular biology. Since its first isolation 1, human umbilical vein endothelial cells ...

Isolation and Culture of Neonatal Mouse Cardiomyocytes

Cultured neonatal cardiomyocytes have long been used to study myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow for easy investigation and manipulation of biochemical pathways, and their effect on the biomechanical properties of spontaneously beating cardiomyocytes.	
The following 2-day protocol describes the isolation and culture of neonatal mouse cardiomyocytes. We show how to easily dissect hearts from neonates, dissociate the cardiac tissue and enrich cardiomyocytes from the cardiac cell-population. We discuss the usage of different enzyme mixes for cell-dissociation, and their effects on cell-viability. The isolated cardiomyocytes can be subsequently used for a variety of morphological, electrophysiological, biochemical, cell-biological or biomechanical assays. We optimized the protocol for robustness and reproducibility, by using only commercially available solutions and enzyme mixes that show little lot-to-lot variability. We also address common problems associated with the isolation and culture of cardiomyocytes, and offer a variety of options for the optimization of isolation and culture conditions.

Primary mouse cardiomyocyte cultures are one of the pivotal tools for the investigation of myofibrillar organization and function. The following protocol describes the isolation and culture of primary cardiomyocytes from neonatal mouse hearts. The resulting cardiomyocyte cultures may be subsequently used for a variety of biomechanical, biochemical and cell-biological assays.

분리 및 문화 신생아 마우스 심근

Cultured neonatal cardiomyocytes have long been used to study  myofibrillogenesis and myofibrillar functions. Cultured cardiomyocytes allow  for easy ...

Isolation of Pulmonary Artery Smooth Muscle Cells from Neonatal Mice

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling pathways involved in vascular smooth muscle contraction in PASMC from fetal sheep. While sheep make an excellent model of term pulmonary hypertension, they are very expensive and lack the advantage of genetic manipulation found in mice. Conversely, the inability to isolate PASMC from mice was a significant limitation of that system. Here we described the isolation of primary cultures of mouse PASMC from P7, P14, and P21 mice using a variation of the previously described technique of Marshall et al.26 that was previously used to isolate rat PASMC. These murine PASMC represent a novel tool for the study of signaling pathways in the neonatal period. Briefly, a slurry of 0.5% (w/v) agarose + 0.5% iron particles in M199 media is infused into the pulmonary vascular bed via the right ventricle (RV). The iron particles are 0.2 &#956;M in diameter and cannot pass through the pulmonary capillary bed. Thus, the iron lodges in the small pulmonary arteries (PA). The lungs are inflated with agarose, removed and dissociated. The iron-containing vessels are pulled down with a magnet. After collagenase (80 U/ml) treatment and further dissociation, the vessels are put into a tissue culture dish in M199 media containing 20% fetal bovine serum (FBS), and antibiotics (M199 complete media) to allow cell migration onto the culture dish. This initial plate of cells is a 50-50 mixture of fibroblasts and PASMC. Thus, the pull down procedure is repeated multiple times to achieve a more pure PASMC population and remove any residual iron. Smooth muscle cell identity is confirmed by immunostaining for smooth muscle myosin and desmin.

We have developed a novel and reproducible technique to isolate primary cultures of pulmonary artery smooth muscle cells (PASMC) from mice as young as P7, thereby allowing better study of the signaling pathways involved in neonatal smooth muscle cell contraction and relaxation.

신생아 쥐의 폐 동맥 평활근 세포의 분리

Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling ...

In Vivo SiRNA Transfection and Gene Knockdown in Spinal Cord via Rapid Noninvasive Lumbar Intrathecal Injections in Mice

This report describes a step-by-step guide to the technique of acute intrathecal needle injections in a noninvasive manner, i.e. independent of catheter implantation. The technical limitation of this surgical technique lies in the finesse of the hands. The injection is rapid, especially for a trained experimenter, and since tissue disruption with this technique is minimal, repeated injections are possible; moreover immune reaction to foreign tools (e.g. catheter) does not occur, thereby giving a better and more specific read out of spinal cord modulation. Since the application of the substance is largely limited to the target region of the spinal cord, drugs do not need to be applied in large dosages, and more importantly unwanted effects on other tissue, as observed with a systemic delivery, could be circumvented1,2. Moreover, we combine this technique with in vivo transfection of nucleic acid with the help of polyethylenimine (PEI) reagent3, which provides tremendous versatility for studying spinal functions via delivery of pharmacological agents as well as gene, RNA, and protein modulators.

In Vivo SiRNA Transfection and Gene Knockdown in Spinal Cord via Rapid Noninvasive Lumbar Intrathecal Injections in Mice

This report describes a simple and rapid technique of intrathecal needle puncture for a localized transfection of siRNA in the lumbar spinal cord in mouse under short lasting light anesthesia.

마우스의 빠른 비 침습 요추 경 막내 주사를 통해 생체 siRNA의 형질과 척수에있는 유전자 넉다운에

This report describes a step-by-step guide to the technique of acute intrathecal needle injections in a noninvasive manner, i.e. independent of catheter ...

Isolation of Neonatal Extrahepatic Cholangiocytes

The intra and extrahepatic bile ducts of the liver are developmentally distinct, and may be differentially affected by certain diseases. However, differences between intra and extrahepatic cholangiocytes, and between neonatal and adult cells, are not well understood.
Methods for the isolation of cholangiocytes from intrahepatic bile ducts are well established1-4. Isolation of extrahepatic ductal cells, especially from the neonate, has not yet been described, although this would be of great benefit in understanding the differences between distinct cholangiocyte populations and in studying diseases such as biliary atresia that appear to target the extrahepatic ducts. Described here is an optimized technique to isolate both neonatal and adult mouse extrahepatic bile duct cells. This technique yields a pure cell population with minimal contamination from mesenchymal cells like fibroblasts.
This method is based on the removal of the extrahepatic ducts and gallbladder, followed by meticulous dissection and scraping to remove fat and fibroblast layers. Structures are embedded in thick layers of collagen and cultured for approximately 3 weeks to allow outgrowth of cholangiocytes in monolayers, which can then be trypsinized and re plated for experimental use.

A technique to isolate cholangiocytes from the extrahepatic bile ducts of neonatal mice is described. The ducts are meticulously dissected, and then cells are isolated by outgrowth in thick collagen gels. This method provides a useful tool for studying extrahepatic bile duct development and pathology.