Авторы признают NINDS, FightSMA и семьям SMA для финансовой поддержки. SEGL поддерживается NINDS учебного гранта # 5T32NS077984-02.

Все процедуры, перечисленные в протоколе были утверждены институт животноводства пользования и комитета Уход (IACUC) из Университета штата Огайо. 1. Подготовка рабочего пространства <ol><li> Соберите мокрый лед, чтобы обезболить щенков мыши, пустую клетку, чтобы отделить плотину из помета, вскрытии микроскопом, источника света, который может быть расположен под углом к ​​инъекции (использование источника света на 90 ° &#160; Угол в месте инъекции затеняет вены), чистую поверхность, чтобы поместить животное для инъекций, ватные тампоны, 3/10 куб.см инсулиновый шприц с 3/8 &quot;30 G иглы (по одному на животных) и 1% синий краситель Эванса ( сделаны с фосфатным буферным солевым раствором (PBS)) раствор для обучения. </li><li> Снимите дамбу в отдельную клетку, манипулируя при щенков. </li></ol> Процедура 2. Инъекции <ol><li> Поместите один щенок прямо на льду в течение 30-60 сек обезболить животное. Не оставляйте тон животное на льду слишком долго из-за риска переохлаждения осложнений, включая фибрилляции желудочков, тканевой гипоксии и метаболического ацидоза. ПРИМЕЧАНИЕ: Наш опыт показывает, что 30-60 сек достаточно, чтобы замедлить движение щенок, чтобы для инъекций. Если глубже анестезия не требуется, ингаляции, такие как 1-2% изофлуран может быть целесообразно. </li><li> В то время как животное на льду, загрузить шприц с 30 мкл Эванса синего красителя. </li><li> Когда животное полностью под наркозом, подтверждается отсутствием движения на льду в то время как все еще дышит, переместите его под микроскопом. Для правосторонней инъекции, сталкиваются морду животного вправо. Поставьте левую указательный палец на морде и левого среднего пальца каудально ушным вкладышем так что ухо бутон между указательным и средним пальцами (рис 1). </li><li> Изучить только впереди ушной зародыше для поверхностного капилляра, что движется, когда кожа манипулируют. Это капиллярной не является целью, однакоидентификация является важным для временной идентификации вен. Затем найдите темный, смутный вену, ниже капилляра, что остается фиксированной, независимо от положения кожи. Временная вены появляется призрачная, работает от спины к брюху, и каналы в яремную вену (рис 1). </li><li> Введите временной вену с иглы конической вверх. Если правильно установлен, его можно просмотреть игла коническая наполняются кровью через кожу. Затем нажмите на поршень медленно и примечание побледнение вену вниз в сторону лица. </li><li> Разрешить игла остается в вене для дополнительного 10-15 сек для предотвращения обратного потока инъекционной. </li></ol> 3. После инъекции <ol><li> После правильного введения, щенок должен почти сразу синеют. Удалите иглу и нежный силу не применять ватный тампон, чтобы в месте инъекции, пока тромбов. </li><li> Монитор щенка на наличие признаков бедствия. Разрешить щенок 2-3 мин, чтобы восстановить и отогреть, гecognize когда щенок находится в сознании, в вертикальном положении и перемещения, прежде чем вернуться в клетку. Кубок щенки в перчатках следователя предоставить соответствующую тепло, чтобы помочь в восстановлении, если это необходимо. Кроме того, место грелку под дом клетке для облегчения прогрева введенного щенка. Вообще после инъекции голубого красителя Эванса, усыпить щенков. Не включать краситель при введении исследуемого материала. </li><li> Поставьте щенка обратно в дом клетке и обеспечить щенок покрыт постельными принадлежностями и / или nestlet обеспечить обратный прием по плотине. </li><li> Используйте новый шприц и ватный тампон для каждого щенка для поддержания стерильности. </li></ol>

<ol>
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Внутрисосудистого поставка агентов в ЦНС или по всему телу трудно в неонатальных мышиных моделях болезни. Описанный протокол является быстрым, относительно неинвазивным способом внутривенно вводить растворы в неонатальном мышей с минимальными требованиями к оборудованию. Хотя врем...

Доставка терапевтических до центральной нервной системы (ЦНС) в мышиных моделях инфекции у детей остается проблемой. Мыши, которые модели новорожденных болезненных состояний являются низкорослыми, а с развитием незрелыми, и, следовательно, может быть трудно вводить непосредственно в соответствующих структур в ЦНС. Внутрисосудистого введения терапевтических агентов является неинвазивным, хорошо переносится метод доставить клетки, наркотики, или вирусные векторы для всего тела, включая ЦНС 1-5 и сетчатки 3,5-9. Предыдущие публикации описывают временную инъекции лицо вен с помощью просвечивания 10,11, без микроскопом рассечение 11,12, или требующих двух лиц, чтобы ввести 10. Техника инъекции описано в данном протоколе выгодно, потому что один человек может придать щенков, и источник света, чтобы просмотреть временная вена не касаясь щенка, устраняя необходимость в хирургической кассете или прикрепление щенка к неподвижной поверхноститакие как просвечивания 11. Доставка аденоассоциированного вирусный вектор серотипа 9 (AAV9) у мышей производит сильную экспрессию в нейронах и астроцитов всему головного и спинного мозга (рис 1). Внутрисосудистого доставка вирусных векторов в поверхностной височной лица вены была надежно использоваться в различных исследованиях в неонатальном мышей лечить педиатрической нервно-мышечной расстройства Спинальная мышечная атрофия (СМА) 2,4,13,14 и в конечном счете увеличивается продолжительность жизни мышей. Внутрисосудистого введения новорожденных мышей также эффективно нацелен на периферическую нервную систему и периферические органы (рис 2). После инъекции AAV, трансдукция ганглиях задних корешков, печени, сердца, скелетной мышце, легких, мышечной оболочки кишечника и сплетения кишечника наблюдалось 1,3,6,7,15. Широкое трансдукция ЦНС и на периферии делает этот метод впрыска идеала для заболеваний, требующих глобального выражениетрансген, таких как болезни 16 и другие хранилища лизосомальных болезней Гоше 17,18, болезни Баттена и связанных с ними нейронов цероид lipofuscinoses, 19 и синдромом Барде-Biedl, генетическое заболевание мультисистемной с момента появления симптомов, возникающих в раннем детстве 20. Внутрисосудистого введения в неонатальном мышей также следует рассматривать в качестве нового метода общесистемных моделирования педиатрических заболеваний. Эта техника была переведена на более крупных животных моделях 5,21 и внутрисосудистой инъекции уже существует в виде клинически приемлемый метод доставки терапевтических. Текущий протокол описывает простой, эффективный метод не доставки агентов на новорожденных мышах через поверхностной височной лица вены не позднее послеродовой день 2. Инъекции могут быть укомплектованы одной, практикуется индивидуальный и хорошо переносится как детенышей и плотин. Щенки испытывать минимальное бедствие и быстро восстанавливаться. ImportanTLY, успешно впрыска приведет к глобальной доставки средства, вводимого. Этот протокол является подходящим для доставки вирусных векторов, фармацевтических агентов или клеток, к новорожденных мышей.

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			<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>
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			<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>
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		<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>
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</table>

intravenous injections in neonatal mice

Внутривенная инъекция является клинически применимо манера доставить терапевтические. Для взрослых грызунов и более крупных животных, внутривенные инъекции, технически осуществимо и рутина. Тем не менее, некоторые модели мышей могут иметь раннее начало заболевания с быстрым прогрессированием, что делает администрация потенциальных методов лечения трудно. Временной (или лица) вена просто передней к ушным вкладышем на мышах и отчетливо видна в течение первых двух дней после родов по обе стороны от головки с использованием рассекает микроскопом. В течение этого окна, временная вены может быть введен с объемами до 50 мкл. Инъекция является безопасным и хорошо переносится как детенышей и плотин. Типичная процедура инъекции завершается в течение 1-2 мин, после чего щенок возвращается к домашней клетке. К третьему день после рождения жила трудно представить себе и процедура инъекции становится технически ненадежны. Эта техника была использована для доставки вируса аденоассоциированный (AAV) VectПРС, что в свою очередь может обеспечить почти тела шириной, стабильную экспрессию трансгена для жизни животного в зависимости от вирусной серотипа выбранной.

Во время правильного инъекции, вены должны на короткое время становится ясным, или бланшировать. Если инъекционных красить весь щенок должен синеть в течение нескольких секунд. Если неправильная инъекция произошла, часто есть концентрированное подкожной болюсной в голову или шею и ин...

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

Intravenous Injections in Neonatal Mice

Животные модели детской болезни могут испытать раннее возникновение и прогрессирование агрессивное заболевание. Клинически значимые доставка терапия для молодых мышах может быть технически сложно. Этот протокол описывает неинвазивный внутривенного метод впрыска для новорожденных мышей в течение первых двух послеродовых дней жизни.

Внутривенных инъекций в новорожденных мышей

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

Research

JoVE Journal

Biology

57.6K Views.  Ohio State University.  Животные модели детской болезни могут испытать раннее возникновение и прогрессирование агрессивное заболевание. Клинически значимые доставка терапия для молодых мышах может быть технически сложно. Этот протокол описывает неинвазивный внутривенного мето?...

Video: 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.

Гетеротопическая трансплантации сердца у мышей

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

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.

Малый трансплантации кишечника у мышей

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.

Поперечные Сужение аорты у мышей

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.

Амбулаторно запись ЭКГ у мышей

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.

Внутривенно микроинъекции данио рерио Личинки по изучению Острое повреждение почек

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.

Выделение и культуре эндотелиальных клеток легочной от новорожденных мышей

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.

Выделение и Культура неонатологическую мыши кардиомиоцитов

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.

Выделение легочной артерии гладкомышечных клеток у новорожденных мышей

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.

В Vivo миРНК трансфекции и Нокдаун гена в спинного мозга через Rapid неинвазивной поясничного Intrathecal инъекций у мышей

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.