وأيد هذا العمل منحة SCCOR &quot;مرض AAA : آليات ، الطبقات ، والمعالجة&quot; (P50 HL083800 - 03) من المعاهد الوطنية للصحة

<ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> الفئران هي تخدير مع استنشاق isoflurane L. / 2.0 - 3.0ml </li><li style=";text-align:right;direction:rtl"> وحلق البطن و تنظيفها بالكحول وbetadine. </li><li style=";text-align:right;direction:rtl"> يتم وضع الماوس مستلق على الحقل المنطوق. </li><li style=";text-align:right;direction:rtl"> يتم ارتداء قفازات معقمة من قبل الجراح ، وجرى تعقيم الأدوات المستخدمة معقمة مع حبة الساخنة. </li><li style=";text-align:right;direction:rtl"> يتم إجراء شق البطن طويلة خط الوسط. وتراجع محتويات البطن خارج البطن مع شاش معقم لفضح الشريان الأورطي البطني. </li><li style=";text-align:right;direction:rtl"> يتم عزل الشريان الأورطي البطني من مستوى الوريد الكلوي الأيسر إلى التشعب. </li><li style=";text-align:right;direction:rtl"> يتعرض لها فروع الشريان الأورطي في البطن وligated مع 10-0 الغرز. و تربط جميع فروع داخل الأبهر 1.0cm من التشعب. </li><li style=";text-align:right;direction:rtl"> توضع الأربطة الحريرية 6.0 مؤقتة حول أجزاء الدانية والقاصية من الشريان الأورطي </li><li style=";text-align:right;direction:rtl"> يتم إنشاء بضع الأبهر في التشعب مع إبرة قياس 30. </li><li style=";text-align:right;direction:rtl"> هو عرض للحرارة أنابيب البولي ايثيلين مدبب (PE - 10) خلال بضع الأبهر والمضمون مع ربطة عنق. </li><li style=";text-align:right;direction:rtl"> باستخدام كيس معلق المالحة في ذروة من 136 سم الى 100 مم زئبق معايرة ، يتم تعبئة الأبهر مع المياه المالحة التي تحتوي على 4.5 U / مل نوع البنكرياس الخنزيري إيلاستاز أنا. الأورطى يتوسع عادة إلى 150-170 ٪ من حجمه الأصلي خلال نضح إيلاستاز 5-15 دقيقة. </li><li style=";text-align:right;direction:rtl"> ثم تتم إزالة القسطرة ونضح بضع الأبهر مغلقة مع خياطة 10-0 لتجنب انقباض. </li><li style=";text-align:right;direction:rtl"> بعد إغلاق بضع الأبهر ، تتم إزالة ربطة القاصي. </li><li style=";text-align:right;direction:rtl"> إذا كان هناك نزيف ضئيلة أو معدومة من بضع الأبهر إصلاحه ، يتم إزالة ربطة القريبة. توسيع الشريان الأورطي يملأ مع دم على الفور. </li><li style=";text-align:right;direction:rtl"> يتم إرجاع الأمعاء إلى البطن ، ويتم إغلاق الجرح في طبقتين مع خاط النايلون / البولي بروبلين 6-0. </li><li style=";text-align:right;direction:rtl"> يتم وضع قطرات Bupivicaime 0.25 ٪ (appx قطرة واحدة لكل سنتيمتر من حجم الجرح) على الجرح وجرعة واحدة من Caprofen (5mg/kg) وتعطى تحت الجلد. </li><li style=";text-align:right;direction:rtl"> يتم وضع الماوس على المنطقة الدافئة ويسترد عادة في غضون 15 دقيقة. </li></ol><img src="/files/ftp_upload/1280/fig1280.png" alt="الشكل 1" /> الشكل 1. النتيجة ممثل إيلاستاز يسببها AAA.

<ol>
	<li>Anidjar, S., Salzmann, J. L., Gentric, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elastase-induced+experimental+aneurysms+in+rats.">Elastase-induced experimental aneurysms in rats.</a> Circulation. 82 (3), 973-973 (1990).</li><li>Pyo, R., Lee, J. K., Shipley, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+disruption+of+matrix+metalloproteinase-9+(gelatinase+B)+suppresses+development+of+experimental+abdominal+aortic+aneurysms.">Targeted gene disruption of matrix metalloproteinase-9 (gelatinase B) suppresses development of experimental abdominal aortic aneurysms.</a> J Clin Invest. 105 (11), 1641-1641 (2000).</li><li>Carsten, C. G., Calton, W. C., Johanning, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elastase+is+not+sufficient+to+induce+experimental+abdominal+aortic+aneurysms.">Elastase is not sufficient to induce experimental abdominal aortic aneurysms.</a> J Vasc Surg. 33 (6), 1255-1255 (2001).</li></ol>

The authors have nothing to disclose.

وقد لاحظت دراسات سابقة هامة الكثير الكثير إلى التباين في التنمية من مختلف التحضيرات AAA إيلاستاز التجاري 3. وبالتالي ، فمن الأهمية بمكان رصد الكثير الإنتاج ، فضلا عن أن جماعات المراقبة المناسبة بغية توثيق القدرة على إعداد للحث على AAAS. ومن المثير للاهتمام ، وقابلية للتنمية AAA في هذا النموذج يعتمد على سلالة تستخدم الماوس 3. الماوس المستخدمة في الفيديو الحالي هو من سلالة C57Bl / 6 الذي وضع ثابت من AAA باستخدام بروتوكول المحددة. ويمكن تعديل تركيز إيلاستاز و / أو الوقت لاستيعاب الحضانة السلالات الأخرى. على الرغم من أن تقنية إنشاء AAA مع إيلاستاز في الفئران تشكل تحديا تقنيا ، وهو أسلوب مهم لدراسة الآليات الجزيئية الكامنة وراء تشكيل AAA والتنمية. وقد أدت الممارسات والتقنية تعديلات في نسبة النجاح أكثر من 90 ٪ في المختبر لدينا. ونحن نعتقد أن الفيديو والنص المصاحب سيساعد في خفض منحنى التعلم للمختبرات التي ترغب في استخدام هذه التقنية. المشاكل <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> وينبغي ligated فروع الشريان الأورطي البطني مع 10-0 خياطة الجروح ، ومنع النزيف أثناء العملية والتوسع غير كافية خلال التسريب إيلاستاز بسبب تسريب المعلومات. </li><li style=";text-align:right;direction:rtl"> فمن المهم أن يكون هناك مجال تشغيل واضحة وتوسعية. ولذلك ، استخدم بدلا من خياطة 6-0 المشابك لانسداد الشريان الأورطي مؤقتا </li><li style=";text-align:right;direction:rtl"> يجب أن يتم مسح تجويف الشريان الأورطي البطني مع محلول ملحي لمنع تجلط الدم بعد العملية. </li><li style=";text-align:right;direction:rtl"> من المهم أن غيض من الحرارة مدبب PE - 10 أنبوب ينبغي على نحو سلس من أجل تجنب خدش الطبقة البطانية عند إدراجها. </li><li style=";text-align:right;direction:rtl"> وينبغي تجنب فقاعات الهواء داخل الأوعية عندما غرس إيلاستاز وكذلك عند إغلاق بضع الأبهر. </li></ol>

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Type I porcine pancreatic elastase</td>
<td>Reagent</td>
<td>Sigma</td>
<td>E1250</td>
<td>&nbsp;</td>
<tr>
<td>polyethylene tubing (PE-10)</td>
<td>&nbsp;</td>
<td>CLAY ADAMS</td>
<td>427400</td>
<td>&nbsp;</td>
<tr>
<td>CHANGE-A-TIP</td>
<td>&nbsp;</td>
<td>Bovie</td>
<td>18010-00 (FST)</td>
<td>cautery knife</td>
</tr>
<tr>
<td>LEICA MZ6</td>
<td>&nbsp;</td>
<td>Leica</td>
<td>&nbsp;</td>
<td>stereomicroscope</td>
</tr>
<tr>
<td>LEICA DFC290HD</td>
<td>&nbsp;</td>
<td>Leica</td>
<td>&nbsp;</td>
<td>USB camera</td>
</tr>
<tr>
<td>Image Pro Express ver. 6.0</td>
<td>&nbsp;</td>
<td>Media Cybernetics</td>
<td>&nbsp;</td>
<td>software</td>
</tr>		</tbody>
		</table>
		</div>

creation of murine experimental abdominal aortic aneurysms with elastase 

التسريب داخل اللمعة عابر إيلاستاز البنكرياس الخنازير في الجزء infrarenal من الشريان الأورطي البطني هو النموذج الحيواني الأكثر استخداما على نطاق واسع من الأبهر البطني (AAA) منذ ذلك الحين وصفت لأول مرة في الفئران عن طريق Anidjar والزملاء ، واستند (1) الأساس المنطقي لتنميتها على طبيعة متقطعة من الإيلاستين التي لوحظت في AAAS. وقد تم تعديل هذا النموذج الفئران لانتاج AAAS في المنطقة الأبهر infrarenal من الفئران. (2) نموذج لديه القدرة على رؤية واسعة إضافة إلى البيولوجيا المرضية من AAA نظرا لظهور العديد من الفئران المعدلة وراثيا خروج المغلوب الجينات. وعلاوة على ذلك ، بل هو منصة قابلة للاختبار وكلاء العلاجية المحتملة لAAA. في هذا الفيديو ، ونحن لشرح إجراءات ضخ إيلاستاز AAA المستخدمة في المختبر لدينا. يتم تخدير الفئران باستخدام 2.5 ٪ isoflurane ، ويتم تنفيذ عملية فتح البطن تحت ظروف معقمة. وaortais البطن المعزولة بمساعدة من stereomicroscope التشغيل (لايكا). بعد وضع الحروف المركبة مؤقتة حول الشريان الأورطي القريبة والبعيدة ، يتم إنشاء بضع الأبهر في التشعب مع غيض من إبرة عيار 30. وقدم قطعة للحرارة مدبب من أنابيب البولي اثيلين PE - 10 خلال بضع الأبهر والمضمون. هو perfused لاحقا لمعة الأبهر 5-15 دقائق في 100 ملم زئبق مع المياه المالحة التي تحتوي على نوع الأول الخنزيري إيلاستاز البنكرياس (4.5 U / مل ؛ سيغما كيميكال). بعد إزالة القسطرة التروية ، يتم إصلاح بضع الأبهر دون انقباض في التجويف.

Watch this Scientific Journal Video about Creation of Murine Experimental Abdominal Aortic Aneurysms with Elastase at JoVE.com

Creation of Murine Experimental Abdominal Aortic Aneurysms with Elastase

هذا الفيديو يبين كيف للحث على تمدد الأوعية الدموية الأبهري البطني (AAA) في الفئران عن طريق التسريب داخل اللمعة عابر إيلاستاز البنكرياس الخنازير في الجزء infrarenal من الشريان الأورطي البطني. نموذج لديه القدرة على رؤية واسعة إضافة إلى البيولوجيا المرضية من AAA نظرا لظهور العديد من الفئران المعدلة وراثيا خروج المغلوب الجينات.

خلق الفئران التجريبية تمدد الأوعية الدموية الأبهري البطني مع إيلاستاز

Transient intraluminal infusion of porcine pancreatic elastase into the infrarenal segment of the abdominal aorta is the most widely used animal model of ...

creation-murine-experimental-abdominal-aortic-aneurysms-with

Nanyang Technological University

Research

JoVE Journal

Biology

Creation of Murine Experimental Abdominal Aortic Aneurysms with Elastase 

24.4K Views.  Stanford University School of Medicine. هذا الفيديو يبين كيف للحث على تمدد الأوعية الدموية الأبهري البطني (AAA) في الفئران عن طريق التسريب داخل اللمعة عابر إيلاستاز البنكرياس الخنازير في الجزء infrarenal من الشريان الأورطي البطني. نموذج لديه القدرة على رؤية واسعة إضافة إلى البيولوجيا المرضية من AAA نظرا لظهو...

Video: Creation of Murine Experimental Abdominal Aortic Aneurysms with Elastase

Aortic Ring Assay

Angiogenesis, the sprouting of blood vessels from preexisting vasculature is associated with both natural and pathological processes. Various angiogenesis assays involve the study of individual endothelial cells in culture conditions (1). The aortic ring assay is an angiogenesis model that is based on organ culture. In this assay, angiogenic vessels grow from a segment of the aorta (modified from (2)). Briefly, mouse thoracic aorta is excised, the fat layer and adventitia are removed, and rings approximately 1 mm in length are prepared. Individual rings are then embedded in a small solid dome of basement matrix extract (BME), cast inside individual wells of a 48-well plate. Angiogenic factors and inhibitors of angiogenesis can be directly added to the rings, and a mixed co-culture of aortic rings and other cell types can be employed for the study of paracrine angiogenic effects. Sprouting is observed by inspection under a stereomicroscope over a period of 6-12 days. Due to the large variation caused by the irregularities in the aortic segments, experimentation in 6-plicates is strongly advised. Neovessel outgrowth is monitored throughout the experiment and imaged using phase microscopy, and supernatants are collected for measurement of relevant angiogenic and anti-angiogenic factors, cell death markers and nitrite.

Angiogenesis, the sprouting of blood vessels from pre-existing vasculature, is associated with both natural and pathological processes. Here we demonstrate an aortic ring assay that allows angiogenic potentiators and inhibitors to be directly added to aortic rings in culture. Sprouting and neovessel outgrowth can be determined by inspecting the aortic rings over a period of 6-12 days.

الأبهر خاتم الفحص

Angiogenesis, the sprouting of blood vessels from preexisting vasculature is associated with both natural and pathological processes. Various angiogenesis ...

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 ...

Isolation and Kv Channel Recordings in Murine Atrial and Ventricular Cardiomyocytes

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their functional properties. Mutations in KCNE genes have been found in patients with cardiac arrhythmias such as the long QT syndrome and/or atrial fibrillation. However, the precise molecular pathophysiology that leads to these diseases remains elusive. In previous studies the electrophysiological properties of the disease causing mutations in these genes have mostly been studied in heterologous expression systems and we cannot be sure if the reported effects can directly be translated into native cardiomyocytes. In our laboratory we therefore use a different approach. We directly study the effects of KCNE gene deletion in isolated cardiomyocytes from knockout mice by cellular electrophysiology - a unique technique that we describe in this issue of the Journal of Visualized Experiments. The hearts from genetically engineered KCNE mice are rapidly excised and mounted onto a Langendorff apparatus by aortic cannulation. Free Ca2+ in the myocardium is bound by EGTA, and dissociation of cardiac myocytes is then achieved by retrograde perfusion of the coronary arteries with a specialized low Ca2+ buffer containing collagenase. Atria, free right ventricular wall and the left ventricle can then be separated by microsurgical techniques. Calcium is then slowly added back to isolated cardiomyocytes in a multiple step comprising washing procedure. Atrial and ventricular cardiomyocytes of healthy appearance with no spontaneous contractions are then immediately subjected to electrophysiological analyses by patch clamp technique or other biochemical analyses within the first 6 hours following isolation.

Kv channel dysfunction is associated with cardiac arrhythmias. In order to study the molecular mechanisms that lead to such arrhythmias we utilize a systematic protocol for isolation of atrial and ventricular cardiomyocytes from Kv channel ancillary subunit knockout mice. Isolated cardiomyocytes can then immediately be used for cellular electrophysiological studies, biochemical or immunofluorescence (IF) assays.

العزلة وتسجيلات قناة كيلو فولت في الفئران Cardiomyocytes الأذيني البطيني و

KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their ...

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca2+ indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca2+ indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca2+ indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca2+ indicator and a hydrophilic fluorescent dye/Ca2+ complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of &Delta;F/F0.

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading TED

Targeted-esterase induced dye loading (TED) supports the analysis of intracellular calcium store dynamics by fluorescence imaging. The method bases on targeting of a recombinant Carboxylesterase to the endoplasmic reticulum (ER), where it improves the local unmasking of synthetic low-affinity Ca2+ indicator dyes in the ER lumen.

التصوير المباشر من الكالسيوم مع ER-المستهدفة استريز المستحثة صبغ تحميل (TED)

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent ...

Isolation and Primary Culture of Mouse Aortic Endothelial Cells

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an important model for cardiovascular disease research. This study demonstrates a simple method to isolate and culture endothelial cells from the mouse aorta without any special equipment. To isolate endothelial cells, the thoracic aorta is quickly removed from the mouse body, and the attached adipose tissue and connective tissue are removed from the aorta. The aorta is cut into 1 mm rings. Each aortic ring is opened and seeded onto a growth factor reduced matrix with the endothelium facing down. The segments are cultured in endothelial cell growth medium for about 4 days. The endothelial sprouting starts as early as day 2. The segments are then removed and the cells are cultured continually until they reach confluence. The endothelial cells are harvested using neutral proteinase and cultured in endothelial cell growth medium for another two passages before being used for experiments. Immunofluorescence staining indicated that after the second passage the majority of cells were double positive for Dil-ac-LDL uptake, Lectin binding, and CD31 staining, the typical characteristics of endothelial cells. It is suggested that cells at the second to third passages are suitable for in vitro and in vivo experiments to study the endothelial biology. Our protocol provides an effective means of identifying specific cellular and molecular mechanisms in endothelial cell physiopathology.

The vascular endothelial cells play a significant role in many important cardiovascular disorders. This article describes a simple method to isolate and expand endothelial cells from the mouse aorta without using any special equipment. Our protocol provides an effective means of identifying mechanisms in endothelial cell physiopathology.

العزلة والثقافة الابتدائية من خلايا الفأر الأورطي غشائي

The vascular endothelium is essential to normal vascular homeostasis. Its dysfunction participates in various cardiovascular disorders. The mouse is an ...

An Alternative Approach for Sample Preparation with Low Cell Number for TEM Analysis

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, especially cells in suspension such as hematopoietic stem cells (HSCs), remains limited due to the requirement of a high cell number during sample preparation. There are a few cytospin or monolayer approaches for TEM analysis from scarce samples, but these approaches fail to get significant quantitative data from the limited number of cells. Here, an alternative and novel approach for sample preparation in TEM studies is described for rare cell populations that enables quantitative analysis.
A relatively low cell number, i.e., 10,000 HSCs, was successfully used for TEM analysis compared to the millions of cells typically used for TEM studies. In particular, Evans blue staining was performed after paraformaldehyde-glutaraldehyde (PFA-GA) fixation to visualize the tiny cell pellet, which facilitated embedding in agarose. Clusters of numerous cells were observed in ultra-thin sections. The cells had a well preserved morphology, and the ultra-structural details of the Golgi complex and several mitochondria were visible. This efficient, easy and reproducible protocol allows sample preparation from a low cell number and can be used for qualitative and quantitative TEM analysis on rare cell populations from limited biological samples.

Here, we present a protocol to prepare samples with low cell numbers for transmission electron microscopy (TEM) analysis.

طريقة بديلة لإعداد نموذج مع انخفاض عدد الخلايا لتحليل تيم

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, ...

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell (VSMC) proliferation. Common murine models study small vessels such as the carotid and femoral arteries. Herein we describe an in vivo system in which both VSMC proliferation and endothelial barrier function can be simultaneously assessed in a large vessel. We studied the infrarenal aortic response to injury in C57BL/6 mice. The aorta was injured from the left renal vein to the aortic bifurcation by 30 transmural crushes of 5-seconds duration with a cotton-tipped applicator. Morphological changes were assessed with conventional histology. Aorta wall thickness was measured from the luminal surface to the adventitia. EdU integration and counter staining with DAPI and alpha-actin was used to demonstrate VSMC proliferation. Activation of ERK1/2, a known moderator of intimal hyperplasia formation, was determined by Western Blot analysis. The effect of inflammation was determined by immunohistochemistry for B-cells, T-cells, and macrophages. En face sections of endothelium were visualized with scanning electron microscopy (SEM). Endothelial barrier function was determined with Evans Blue staining. Transmural injury resulted in aortic wall thickening. This injury induced VSMC proliferation, most prominently at 3 days after injury, and early activation of ERK1/2 and decreased p27kip1 expression. Injury did not result in increased B-cells, T-cells, or macrophages infiltration in the vessel wall. Injury caused partial endothelial cell denudation and loss of cell-cell contact. Injury resulted in a significant loss of endothelial barrier function, which returned to baseline after seven days. The murine transmural blunt aortic injury model provides an efficient system to simultaneously study both VSMC proliferation and endothelial barrier function in a large vessel.

Murine Aortic Crush Injury: An Efficient In Vivo Model of Smooth Muscle Cell Proliferation and Endothelial Function

Restenosis following cardiovascular procedures (bypass surgery, angioplasty, or stenting) is a significant problem reducing the durability of these procedures. An ideal therapy would inhibit smooth muscle cell (VSMC) proliferation while promoting regeneration of the endothelium. We describe a model for simultaneous assessment of VSMC proliferation and endothelial function in vivo.

الفئران الأرواح سحق إصابة: كفاءة<em&gt; في فيفو</em&gt; نموذج من انتشار الخلايا العضلية السلس والوظيفة البطانية

Arterial reconstruction, whether angioplasty or bypass surgery, involves iatrogenic trauma causing endothelial disruption and vascular smooth muscle cell ...

Establishment and Genetic Manipulation of Murine Hepatocyte Organoids

The liver is the largest organ in mammals. It plays an important role in glucose storage, protein secretion, metabolism and detoxification. As the executor for most of the liver functions, primary hepatocytes have limited proliferating capacity. This requires the establishment of ex vivo hepatocyte expansion models for liver physiological and pathological research. Here, we isolated murine hepatocytes by two steps of collagenase perfusion and established a 3D organoid culture as the 'mini-liver' to recapitulate cell-cell interactions and physical functions. The organoids consist of heterogeneous cell populations including progenitors and mature hepatocytes. We introduce the process in detailed to isolate and culture the murine hepatocytes or fetal hepatocyte to form organoids within 2-3 weeks and show how to passage them by mechanically pipetting up and down. In addition, we will also introduce how to digest the organoids into single cells for lentivirus infection of shRNA/ectopic construction, siRNA transfection and CRISPR-Cas9 engineering. The organoids can be used for drug screens, disease modelling, and basic liver research by modeling liver biology and pathobiology.

A long-term ex vitro 3D organoid culture system was established from mouse hepatocytes. These organoids can be passaged and genetically manipulated by lentivirus infection of shRNA/ectopic construction, siRNA transfection and CRISPR-Cas9 engineering.

إنشاء والتلاعب الجيني من الأورجانويدات مورين الكبدية

The liver is the largest organ in mammals. It plays an important role in glucose storage, protein secretion, metabolism and detoxification. As the ...

Application of Passive Head Motion to Generate Defined Accelerations at the Heads of Rodents

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular mechanisms behind the beneficial effects of exercise are poorly understood. Many physical workouts, particularly those classified as aerobic exercises such as jogging and walking, produce impulsive forces at the time of foot contact with the ground. Therefore, it was speculated that mechanical impact might be implicated in how exercise contributes to organismal homeostasis. For testing this hypothesis on the brain, a custom-designed ''passive head motion'' (hereafter referred to as PHM) system was developed that can generate vertical accelerations with controlled and defined magnitudes and modes and reproduce mechanical stimulation that might be applied to the heads of rodents during treadmill running at moderate velocities, a typical intervention to test the effects of exercise in animals. By using this system, it was demonstrated that PHM recapitulates the serotonin (5-hydroxytryptamine, hereafter referred to as 5-HT) receptor subtype 2A (5-HT2A) signaling in the prefrontal cortex (PFC) neurons of mice. This work provides detailed protocols for applying PHM and measuring its resultant mechanical accelerations at rodents' heads.

The present protocol describes a custom-designed ''passive head motion'' system, which reproduces mechanical accelerations at rodents' heads generated during their treadmill running at moderate velocities. It allows dissecting mechanical factors/elements from the beneficial effects of physical exercise.

تطبيق حركة الرأس السلبية لتوليد تسارع محدد عند رؤوس القوارض

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular ...

Isolation of Primary Patient-specific Aortic Smooth Muscle Cells and Semiquantitative Real-time Contraction Measurements In Vitro

Smooth muscle cells (SMCs) are the predominant cell type in the aortic media. Their contractile machinery is important for the transmission of force in the aorta and regulates vasoconstriction and vasodilation. Mutations in genes encoding for the SMC contractile apparatus proteins are associated with aortic diseases, such as thoracic aortic aneurysms. Measuring SMC contraction in vitro is challenging, especially in a high-throughput manner, which is essential for screening patient material. Currently available methods are not suitable for this purpose. This paper presents a novel method based on electric cell-substrate impedance sensing (ECIS). First, an explant protocol is described to isolate patient-specific human primary SMCs from aortic biopsies and patient-specific human primary dermal fibroblasts for the study of aortic aneurysms. Next, a detailed description of a new contraction method is given to measure the contractile response of these cells, including the subsequent analysis and suggestion for comparing different groups. This method can be used to study the contraction of adherent cells in the context of translational (cardiovascular) studies and patient and drug screening studies.

Isolation of Primary Patient-specific Aortic Smooth Muscle Cells and Semiquantitative Real-time Contraction Measurements In Vitro

This paper describes an explant culture-based method for the isolation and culturing of primary, patient-specific human aortic smooth muscle cells and dermal fibroblasts. Furthermore, a novel method is presented for measuring cell contraction and subsequent analysis, which can be used to study patient-specific differences in these cells.

عزل خلايا العضلات الملساء الأبهرية الأولية الخاصة بالمريض وقياسات الانقباض شبه الكمية في الوقت الفعلي في المختبر

Smooth muscle cells (SMCs) are the predominant cell type in the aortic media. Their contractile machinery is important for the transmission of force in ...