الكتاب نود أن نعترف NINDS، FightSMA، وأسر SMA للدعم المالي. ويدعم سيجل من خلال التدريب NINDS منحة # 5T32NS077984-02.

وقد تمت الموافقة على جميع الإجراءات الواردة في بروتوكول معهد للاستخدام الحيواني والعناية جنة (IACUC) من جامعة ولاية أوهايو. 1. إعداد مساحة العمل <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> جمع الجليد الرطب لتخدير الجراء الماوس، قفص فارغ للفصل بين السد من القمامة، ومجهر تشريح، مصدر الضوء الذي يمكن وضعه في زاوية لحقن (استخدام مصدر الضوء في 90 ° &#160; زاوية لتحجب موقع الحقن في الوريد)، سطح نظيف لوضع الحيوان للحقن، ومسحات القطن، 10/03 سم مكعب حقنة الأنسولين مع 3/8 &quot;30 G إبرة (واحد لكل حيوان) و 1٪ ايفانز الأزرق صبغ ( مصنوعة من الفوسفات مخزنة المالحة (PBS)) حل للتدريب. </li><li style=";text-align:right;direction:rtl"> إزالة السد إلى قفص منفصل بينما التلاعب الجراء. </li></ol> 2. إجراء حقن <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> وضع الجرو واحد مباشرة على الجليد الرطب لمدة 30-60 ثانية لتخدير الحيوان. لا تترك رانه حيوان على الجليد فترة طويلة جدا بسبب خطر حدوث مضاعفات انخفاض حرارة الجسم ذات الصلة بما في ذلك الرجفان البطيني، نقص الأكسجة الأنسجة والحماض الأيضي. ملاحظة: تجربتنا هو أن 30-60 ثانية كافية لإبطاء الحركات الجرو للسماح للحقن. إذا كان مطلوبا التخدير أعمق، المستنشقات مثل 1-2٪ الأيزوفلورين قد يكون مناسبا. </li><li style=";text-align:right;direction:rtl"> في حين أن الحيوان هو على الجليد، تحميل المحاقن مع 30 ميكرولتر من ايفانز صبغة زرقاء. </li><li style=";text-align:right;direction:rtl"> عندما يتم تخدير الحيوان بالكامل، والتي أكدتها قلة الحركة على الجليد في حين لا يزال يتنفس، نقله تحت المجهر. لحقن يمنى، تواجه كمامة الحيوان إلى اليمين. وضع السبابة اليسار على كمامة وترك الذيلية الاصبع الوسطى إلى الأذن برعم بحيث برعم الأذن بين السبابة والوسطى (الشكل 1). </li><li style=";text-align:right;direction:rtl"> دراسة فقط الأمامي لبرعم الأذن لسطحية الشعرية التي تتحرك عندما يتم التلاعب الجلد. هذه الشعرية ليس هو الهدف، ولكنتحديد مهم لتحديد الوريد الصدغي. المقبل، وتحديد مكان مظلم، غامض أدنى من الوريد الشعرية التي تظل ثابتة بغض النظر عن الموقف الجلد. يظهر الوريد الصدغي غامضة، يعمل على ظهري بطني، ويغذي في الوريد الوداجي (الشكل 1). </li><li style=";text-align:right;direction:rtl"> أدخل الوريد الصدغي مع الإبرة شطبة تصل. في حالة إدخالها بشكل صحيح، فمن الممكن لعرض شطبة إبرة ملء مع الدم من خلال الجلد. ثم تضغط على المكبس ببطء ومذكرة ابيضاض الوريد أسفل الجانب من وجهه. </li><li style=";text-align:right;direction:rtl"> السماح الإبرة بالبقاء داخل الوريد لمدة 10-15 ثانية مضافة لمنع ارتجاعي من injectant. </li></ol> 3. بعد الحقن <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> بعد الحقن السليم، يجب أن الجرو تتحول الأزرق على الفور تقريبا. إزالة الإبرة واستخدام القوة لطيف لتطبيق مسحة القطن إلى موقع الحقن حتى تجلط الدم. </li><li style=";text-align:right;direction:rtl"> مراقبة الجرو لعلامات الضيق. السماح الجرو 2-3 دقائق لاستعادة وrewarm، صecognize عندما الجرو هو واعية، وتستقيم وتتحرك، قبل أن يعود إلى القفص. فنجان الجراء في أيدي القفاز المحقق لتوفير الدفء المناسب للمساعدة في التعافي إذا لزم الأمر. بدلا من ذلك، وضع وسادة التدفئة تحت القفص المنزل لتسهيل ارتفاع درجة حرارة الجرو حقن. عموما بعد حقن صبغة زرقاء ايفانز، الموت ببطء الجراء. لا تشمل صبغة عند حقن مادة الاختبار. </li><li style=";text-align:right;direction:rtl"> وضع الجرو مرة أخرى في قفص المنزل وضمان المغلفة الجرو مع الفراش و / أو nestlet لضمان reacceptance من قبل السد. </li><li style=";text-align:right;direction:rtl"> استخدام حقنة جديدة والقطن المسحة لكل الجرو للحفاظ على العقم. </li></ol>

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تسليم داخل الأوعية الدموية وكلاء لالجهاز العصبي المركزي أو في جميع أنحاء الجسم أمر صعب في نماذج الفئران حديثي الولادة من الأمراض. بروتوكول صفها هو، طريقة سريعة نسبيا غير الغازية لإدارة حلول عن طريق الوريد في الفئران حديثي الولادة مع متطلبات الحد الأدنى من المعدات. ع...

تقديم العلاجات للجهاز العصبي المركزي (CNS) في نماذج الفئران من أمراض الأطفال ما زال يمثل تحديا. الفئران هذا النموذج الحالات المرضية حديثي الولادة هي الأصغر وغير ناضجة تنمويا، وبالتالي يمكن أن يكون صعبا لحقن مباشرة في الهياكل المناسبة داخل الجهاز العصبي المركزي. الحقن داخل الأوعية الدموية من العوامل العلاجية هو غير الغازية أو أسلوب جيد التحمل لتوصيل الخلايا، والمخدرات، أو ناقلات فيروسية لكامل الجسم بما في ذلك الجهاز العصبي المركزي وشبكية العين 1-5 3،5-9. المنشورات السابقة تصف الزمانية الوريد حقن الوجه باستخدام نقل transilluminator 10،11، دون مجهر تشريح 11،12، أو التي تتطلب شخصين لحقن 10. تقنية الحقن الموصوفة في هذا البروتوكول هو مفيد لشخص واحد يمكن أن تضخ الجراء، ومصدر الضوء لعرض الوريد الصدغي لا لمس الجرو، مما يلغي الحاجة إلى الشريط الجراحية أو مرفق من الجرو على سطح ثابتمثل نقل transilluminator 11. تسليم الغدة المرتبطة ناقلات فيروسية المصلي 9 (AAV9) في الفئران ينتج التعبير القوي في الخلايا العصبية والخلايا النجمية في جميع أنحاء الدماغ والحبل الشوكي (الشكل 1). تسليم داخل الأوعية ناقلات فيروسية في الوريد الوجهي الصدغي السطحي قد استخدمت بشكل صحيح في مختلف الدراسات في الفئران حديثي الولادة لعلاج الخلل العصبي العضلي للأطفال ضمور العضلات الشوكي (SMA) 2،4،13،14 وزادت في نهاية المطاف حياة الفئران المعالجة. الحقن داخل الأوعية الدموية للفئران حديثي الولادة تستهدف أيضا بشكل فعال على الجهاز العصبي المحيطي والأجهزة الطرفية (الشكل 2). بعد حقن AAV، وقد لوحظ تنبيغ العقد الجذرية الظهرية والكبد والقلب والعضلات والهيكل العظمي والرئة، والضفيرة العضلية المعوية للامعاء 1،3،6،7،15. التنبيغ واسع النطاق للجهاز العصبي المركزي ومحيط يجعل هذه الطريقة من الحقن مثالية للأمراض التي تتطلب التعبير العالميالتحوير، مثل مرض 16 وغيرها من أمراض التخزين الليزوزومية غوشر 17،18، مرض باتن وlipofuscinoses سيرويد الخلايا العصبية ذات الصلة، 19 ومتلازمة بارديه-بيدل، وهو اضطراب وراثي المتعددة للمع ظهور الأعراض التي تحدث في مرحلة الطفولة المبكرة (20). وينبغي أيضا النظر الحقن داخل الأوعية الدموية في الفئران حديثي الولادة كوسيلة رواية النمذجة أمراض الأطفال على نطاق المنظومة. وقد ترجمت هذه التقنية لنماذج حيوانية أكبر 5،21، والحقن داخل الأوعية موجود بالفعل كوسيلة مقبولة سريريا لتقديم العلاجات. يصف البروتوكول الحالي وسيلة بسيطة وفعالة لتقديم وكلاء لالفئران حديثي الولادة عن طريق الوريد الصدغي السطحي وجه في موعد أقصاه يوم بعد الولادة 2. حقن يمكن أن يتم الانتهاء من واحدة، يمارس الفرد وجيد التحمل من قبل كل من الجراء والسدود. الجراء تجربة الحد الأدنى من الشدة والتعافي بسرعة. الاهميهTLY، وحقن الناجح يؤدي إلى تسليم العالمي للعامل تدار. هذا البروتوكول هو المناسب لتسليم ناقلات فيروسية، وكلاء الصيدلانية أو خلايا الفئران حديثي الولادة.

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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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		<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>
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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>
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intravenous injections in neonatal mice

الحقن في الوريد هي الطريقة المعمول بها سريريا لتقديم العلاجات. للقوارض الكبار والحيوانات الكبيرة، والحقن الوريدية هي مجدية تقنيا والروتينية. ومع ذلك، يمكن لبعض نماذج الماوس يكون بداية مبكرة من المرض مع التقدم السريع الذي يجعل إدارة العلاجات المحتملة صعوبة. الوريد الصدغي (أو الوجه) هو مجرد الأمامي لبرعم الأذن لدى الفئران وبشكل واضح للعيان اليومين الأولين بعد الولادة على جانبي الرأس باستخدام مجهر تشريح. خلال هذا الإطار، يمكن حقنه في الوريد الصدغي مع وحدات التخزين ما يصل إلى 50 ميكرولتر. الحقن هو آمن وجيد التحمل من قبل كل من الجراء والسدود. اكتمال إجراء حقن نموذجي في غضون 1-2 دقائق، وبعد ذلك يتم إرجاع الجرو إلى قفص المنزل. في اليوم الثالث بعد الولادة الوريد من الصعب تصور ويصبح إجراء الحقن لا يمكن الاعتماد عليها من الناحية الفنية. وقد استخدمت هذه التقنية لإيصال فيروس الغدة المرتبطة (AAV) vectأملاح الإماهة الفموية، والتي بدورها يمكن أن توفر تقريبا في جميع أنحاء الجسم، ومستقر التعبير التحوير لحياة الحيوان اعتمادا على المصلي الفيروسي الذي تم اختياره.

خلال حقن الصحيح، ينبغي أن تتحول الوريد للحظات واضح، أو بلانش. إذا حقن صبغ الجرو كله يجب أن يتحول الأزرق في غضون ثوان. إذا حدث حقنة غير لائق، غالبا ما يكون هناك بلعة تحت الجلد تتركز في الرأس أو الرقبة وinjectant قد يتسرب من موقع الحقن. قد ينتج عن الحقن غير لائقة أيضا في ظهور ك...

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

Intravenous Injections in Neonatal Mice

نماذج حيوانية من المرض لدى الأطفال يمكن أن تواجه بداية مبكرة وتطور المرض العدواني. سريريا تقديم العلاج ذات الصلة لنماذج الماوس الشباب يمكن أن يكون صعبا من الناحية الفنية. يصف هذا البروتوكول طريقة الحقن في الوريد غير الغازية عن الفئران حديثي الولادة في غضون شهرين بعد الولادة الأيام الأولى من الحياة.

الحقن في الوريد في الفئران حديثي الولادة

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.  نماذج حيوانية من المرض لدى الأطفال يمكن أن تواجه بداية مبكرة وتطور المرض العدواني. سريريا تقديم العلاج ذات الصلة لنماذج الماوس الشباب يمكن أن يكون صعبا من الناحية الفنية. يصف هذا البروتوكول طريقة الحقن في الوريد غير الغازية عن الفئران حدي?...

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.

منتبذ زرع القلب في الفئران

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.

زرع الأمعاء الصغيرة في الفئران

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.

المستعرضة في انقباض الأورطي الفئران

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.

الإسعافية ECG التسجيل في الفئران

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.

في الوريد من يرقات اسماك الزرد Microinjections لدراسة إصابة الكلى الحاد

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.

العزلة والثقافة في الخلايا البطانية الرئوي من الفئران حديثي الولادة

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.

العزلة وثقافة الأطفال حديثي الولادة ماوس العضلية

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.

عزل الشريان الرئوي خلايا العضلات الملساء من الفئران حديثي الولادة

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.

في فيفو سيرنا ترنسفكأيشن والجينات ضربة قاضية في الحبل الشوكي عن طريق السريع موسع قطني داخل القراب الحقن في الفئران

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.