作者要感谢SMA的NINDS，FightSMA和家庭提供财政支持。 SEGL由NINDS培训资助＃5T32NS077984-02支持。

在协议中列出的所有程序已经批准学院为美​​国俄亥俄州立大学动物使用及护理委员会（IACUC）。 1.准备工作区<ol><li>收集湿冰麻醉的小鼠幼仔，​​一个空的保持架，以分隔从垫料，在解剖显微镜，其可被定位成一定角度喷射（使用光源以90°的光源坝&#160;角向注射部位掩盖了静脉），清洁表面放置动物的注射，棉签，3/10毫升的胰岛素注射器用3/8“为30G针（每动物1）和1％伊文思蓝染料（与用于训练的磷酸盐缓冲盐水（PBS）中）的溶液制成。 </li><li>删除大坝到一个单独的笼子，而操控幼崽。 </li></ol> 2.注射过程<ol><li>直接将单小狗在湿冰30-60秒，以麻醉的动物。不要离开吨他的动物冰上过长因体温过低相关并发症，包括心室颤动，组织缺氧和代谢性酸中毒的风险。 注：我们的经验是30-60秒，足以减缓小狗的动作，让注射。如果更深麻醉需要，吸入剂，如1-2％异氟醚可以是合适的。 </li><li>而动物是冰，装入注射器30微升的伊文思蓝染料。 </li><li>当动物被完全麻醉，通过在仍呼吸冰缺乏运动的确认后，将它在显微镜下。为右旋注入，面对动物的枪口右侧。将左手的食指上枪口和左手的中指尾椎到耳芽，使耳芽是食指和中指（ 图1）之间。 </li><li>检查只是前耳芽的浅表毛细血管的运动时，皮肤被操纵。该毛细管是不是目标，但是识别是颞静脉识别非常重要。接下来，找到一个黑暗，朦胧的静脉不如保持不变，无论皮肤位置的毛细血管。颞静脉出现神出鬼没，运行背侧到腹侧，并馈入颈静脉（ 图1）。 </li><li>输入与针头斜面向上颞静脉。如果正确地插入时，也能够查看针头斜面充满血液通过皮肤。然后压下柱塞慢慢地和音符静脉向下的面一侧的热烫。 </li><li>允许针留在静脉内用于增加10-15秒，以防止注入剂的回流。 </li></ol> 3.后喷射<ol><li>正确的注射后，小狗应立即变成蓝色。取下针头和使用轻柔的力向棉棒适用于注射部位直到血块。 </li><li>监控小狗遇险的迹象。让小狗2-3分钟，以恢复和复温河ecognize当小狗是有意识的，正直和移动，久违的笼子前。世界杯研究者的戴手套的手幼仔提供适当的保暖，如果有必要，以帮助恢复。或者，将笼下一个加热垫，以方便升温注入小狗。一般的伊文思蓝染料注射后，安乐死幼仔。注射试验材料时，不要包括染料。 </li><li>将小狗放回笼子，确保小狗涂有被褥和/或nestlet确保reacceptance由大坝。 </li><li>使用新的注射器和棉棒每个小狗保持无菌。 </li></ol>

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作者什么都没有透露。

血管内递送药物到中枢神经系统或整个身体难以在疾病的新生鼠模型。所描述的协议，是静脉内给药溶液加入到新生小鼠以最小的设备要求一种快速，相对非侵入性的方式。虽然颞面部静脉可以通过肉眼观察，注射液可以具有更高的准确性与使用显微镜和光纤光源的，特别是对于一个不熟练的喷射器。在新生小鼠的血管内注射剂具有较高的成功率12，和AAV转导已经在早幼崽被观察到即使注...

递送治疗剂到在儿科疾病的鼠模型中的中枢神经系统（CNS）中的仍然是一个挑战。小鼠模型中的新生疾病状态是矮小和发育不成熟，因此可能难以直接注入在中枢神经系统内的适当的结构。血管内注射的治疗剂是一种非侵入性的，良好耐受的方法，提供的细胞，药物，或病毒载体的整个身体，包括中枢神经系统1-5和视网膜3,5-9。以前的出版物描述了使用透10,11颞面部静脉注射，无需解剖镜下11,12，还是需要两个人来注入10。在这个协议中所描述的注射技术是有利的，因为一个人可以注入幼仔，和光源，以查看颞静脉没有触摸小狗，省去了手术带或小狗的连接到一个固定的表面诸如透射11。输送的腺相关病毒载体的血清型9（AAV9）在小鼠中产生强大的表达在神经元和星形胶质细胞的整个大脑和脊髓（ 图1）。血管内递送病毒载体到颞浅面静脉已被可靠地使用在各种研究中新生小鼠治疗小儿神经肌肉病脊髓性肌萎缩（SMA），2,4,13,14，并最终增加了处理的小鼠的寿命。 血管内注射新生小鼠也有效地靶向外周神经系统和外周器官（ 图2）。以下注射的AAV，背根神经节，肝脏，心脏，骨骼肌，肺和肠的肠肌丛的转导已观察1,3,6,7,15。中枢神经系统和周围的广泛传导，使理想的注射这种方法需要对全球的疾病表现转基因，比如高雪氏病16和其他溶酶体贮积病17,18，巴滕病和相关的神经元蜡样lipofuscinoses，19和巴比二氏综合征，一种遗传性多系统疾病发病的早期儿童20出现的症状。血管内注射到新生小鼠中，也应考虑作为新的建模系统范围的儿科疾病的方法。这种技术已被翻译为较大的动物模型5,21和血管内注射已经存在作为递送治疗剂的临床上可接受的方法。 目前的协议描述了通过颞浅静脉脸上交付代理商新生小鼠不晚于产后第2天注射一种简单，有效的方法，可以通过一个单一的完成，实行个人和既受两个幼崽和水坝的耐受性。幼崽体验最小痛苦和快速恢复。 ImportanTLY，成功的注入将导致管理代理的全球交付。这个协议是适当的递送病毒载体，药物制剂或细胞对新生小鼠。

<table border="0" cellpadding="0" cellspacing="0" width="625">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:103px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:188px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Thinpro Insulin Syringe</td>
			<td style="width:103px;">Terumo</td>
			<td>SS30M3009</td>
			<td style="width:188px;">3/10cc, 3/8&#34; needle, 30G, 1 per mouse</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Evans Blue Dye</td>
			<td style="width:103px;">Sigma-Aldrich</td>
			<td style="width:119px;">E2129</td>
			<td style="width:188px;">Dilute to 1% with 1X Phosphate Buffered Saline&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cotton Tipped Applicators</td>
			<td style="width:103px;">Fisher Scientific&#160;</td>
			<td style="width:119px;">23-400-101</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Fiber Optic Light Source&#160;</td>
			<td style="width:103px;">Fisher Scientific&#160;</td>
			<td style="width:119px;">12-562-36</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dissecting Microscope</td>
			<td style="width:103px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

intravenous injections in neonatal mice

静脉注射是一种临床应用的方式提供治疗。对于成年啮齿类动物和大型动物，静脉注射在技术上是可行的程序。然而，一些小鼠模型可以有早发性疾病具有病情进展快，使潜在疗法的管理难度。的时间（或面部）静脉只是前耳芽在小鼠和显然对于前两天可见在出生后的头使用解剖显微镜的任一侧。在此窗口中，颞静脉可以与体积可达50微升注射。注射是安全的，由两个幼仔和堤坝的耐受性良好。一种典型的注射过程中1-2分钟，在此之后，小狗返回到笼中完成。到了第三日龄静脉是很难想象和注射过程在技术上变得不可靠。这一技术已被用于递送的腺相关病毒（AAV）VECT口服补液盐，这反过来又可以用于根据所述病毒血清型所选择的动物的生活提供几乎全身性的，稳定的转基因表达。

在适当的注射，静脉应暂时关闭清楚，或者焯烫。如果注射染料整个小狗应该在几秒钟内变成蓝色。如果发生了不正确的喷射，往往有一个浓缩皮下推注的头部或颈部和注入剂可能泄漏的注射部位的出。不当注射剂也可能会导致围绕喉部青紫的外观。接收皮下注射（ 即注射液没有充分的静脉交付），一般遇到任何不良副作用和常规响应处理的正常小鼠注射做幼仔。 根?...

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

Intravenous Injections in Neonatal Mice

儿科疾病的动物模型可以体验早期发病和严重的疾病进展。临床相关治疗交付给年轻小鼠模型可以在技术上难度。这个协议描述了一种非侵入性的静脉内注射的方法在生命的最初2日龄新生小鼠。

在新生小鼠静脉注射

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

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Biology

57.6K 次观看.  Ohio State University. 儿科疾病的动物模型可以体验早期发病和严重的疾病进展。临床相关治疗交付给年轻小鼠模型可以在技术上难度。这个协议描述了一种非侵入性的静脉内注射的方法在生命的最初2日龄新生小鼠。 

视频: 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.

小鼠异位心脏移植

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

科研

生物学

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.

小鼠小肠移植

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.

在小鼠体内的横向主动脉缩窄

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.

在小鼠体内的动态心电图记录

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.

斑马鱼幼虫的研究急性肾损伤的静脉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.

从新生小鼠的肺血管内皮细胞的分离和培养

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.

分离新生小鼠心肌文化和

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.

肺动脉平滑肌细胞从新生小鼠隔离

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.

体内转染和基因敲除在脊髓通过快速无创腰椎鞘内注射小鼠

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.