Name: focal ca2+ transient detection in smooth muscle
Uploaded: 2009-06-29T00:00:00
Duration: 17 min 41 s
Description: Watch this Scientific Journal Video about Focal Ca2+ Transient Detection in Smooth Muscle at JoVE.com

Wellcome Trust оказывает финансовую поддержку (VIP Премия JSY и 074128 исследовательский грант на КЛБ). Совет по медицинским исследованиям Студенчество в RJA Leica_SP2_Stacker, алгоритм, используемый для импорта Формат TIFF для «реконструкции» в серии, основана на Leica_tiff_sequence, плагин для чтения Leica SP2 Формат TIFF в ImageJ, разработанный Тони Коллинз и используются с его разрешения. ( <a href="http://www.uhnres.utoronto.ca/facilities/wcif/software/Plugins/LeicaTIFF.html">http://www.uhnres.utoronto.ca/facilities/wcif/software/Plugins/LeicaTIFF.html</a> ) StackReg и TurboReg, алгоритмы, используемые для корректировки движения ткани, основаны на Th venaz и коллеги (1998) 9. Excel_writer.jar был написан Куртом Де Вос (http://rsb.info.nih.gov/ij/plugins/excel-writer.html). JNCT0.08JoVE.ijm, алгоритм регистрации частиц, основан на алгоритме написана KL мозга и описаны ранее 5.

<ol>
	<li>Haugland, R. P., Gregory, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Indicators+for+Ca2+,+Mg2+,+Zn2++and+other+metal+ions.">Indicators for Ca2+, Mg2+, Zn2+ and other metal ions.</a> Handbook of fluorescent probes and research products. , 771-826 (2002).</li><li>Boland, B., Himpens, B., Gillis, J. M., Casteels, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+between+force+and+Ca2++in+anococcygeal+and+vas+deferens+smooth+muscle+cells+of+the+mouse.">Relationship between force and Ca2+ in anococcygeal and vas deferens smooth muscle cells of the mouse.</a> Pflugers Arch. 421, 43-51 (1992).</li><li>Hashitani, H., Fukuta, H., Takano, H., Klemm, M. F., Suzuki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin+and+propagation+of+spontaneous+excitation+in+smooth+muscle+of+the+guinea-pig+urinary+bladder.">Origin and propagation of spontaneous excitation in smooth muscle of the guinea-pig urinary bladder.</a> J Physiol. 150, 273-286 (2001).</li><li>Heppner, T. J., Bonev, A. D., Nelson, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elementary+purinergic+Ca2++transients+evoked+by+nerve+stimulation+in+rat+urinary+bladder+smooth+muscle.">Elementary purinergic Ca2+ transients evoked by nerve stimulation in rat urinary bladder smooth muscle.</a> J. Physiol. 564, 201-212 (2005).</li><li>Brain, K. L., Jackson, V. M., Trout, S. J., Cunnane, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intermittent+ATP+release+from+nerve+terminals+elicits+focal+smooth+muscle+Ca2++transients+in+mouse+vas+deferens.">Intermittent ATP release from nerve terminals elicits focal smooth muscle Ca2+ transients in mouse vas deferens.</a> J. Physiol. 541, 849-862 (2002).</li><li>Fleichman, M., Schneider, T., Fetscher, C., Michel, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+transduction+underlying+carbachol-induced+contraction+of+rat+urinary+bladder+II.+Protein+kinases.">Signal transduction underlying carbachol-induced contraction of rat urinary bladder II. Protein kinases.</a> J. Pharmacol. Exp. Ther. 308, 54-58 (2004).</li><li>Young, J. S., Meng, E., Cunnane, T. C., Brain, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+purinergic+neurotransmission+in+the+mouse+urinary+bladder.">Spontaneous purinergic neurotransmission in the mouse urinary bladder.</a> J. Physiol. 586, 5743-5755 (2008).</li><li>Nelson, M. T., Cheng, H., Rubart, M., Santana, L. F., Bonev, A. D., Knot, H. J., Lederer, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relaxation+of+arterial+smooth+muscle+by+calcium+sparks.">Relaxation of arterial smooth muscle by calcium sparks.</a> Science. 270, 633-637 (1995).</li><li>Th&#233;venaz, P., Ruttimann, U. E., Unser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pyramid+approach+to+subpixel+registration+based+on+intensity.">A pyramid approach to subpixel registration based on intensity.</a> IEEE Trans. Image Process. 7, 27-41 (1998).</li></ol>

Были предприняты усилия, чтобы минимизировать число животных, используемых и их страдания, и все эксперименты были проведены в соответствии с животным Великобритании (Научно процедуры) Закона 1986 г. и Европейского Сообщества Директивы Совета 86/09/EEC.

Выбор флуоресцентных Са 2 + индикатор Орегон Зеленый 488 BAPTA-1 утра был выбран в качестве Са 2 + индикатор, поскольку он (я) является яркой по сравнению с другими показателями (например, Fluo-4 и кальция зеленый) 1, (II) является чувствительным к физиологическим изменениям в концентрации Са 2 + с К г такого же масштаба 1 к внутриклеточной концентрации Са 2 + [Са 2 +] я (например, семявыносящих протоков 2); (III) загружает много гладких мышечных клеток, что позволяет гладко мышечных клеток связи для мониторинга. Тем не менее, Орегон Зеленый 488 BAPTA-1 АМ без ratioable Са 2 + индикатор, и как таковые не могут быть легко использованы для определения абсолютных [Са 2 +] я. Кроме того, она может также буфера Са 2 + при наличии в высоких концентрациях и насыщается высокими изменения амплитуды [Ca 2 +] я. Подавление спонтанной сократительной Спонтанной сократительной способности гладких мышц органов может быть уменьшена фармакологически, таких как, блокируя движение Са 2 + через L-типа Ca 2 +-каналов (например, путем: нифедипин 3; дилтиазем 4). Обратите внимание, что эти препараты значительно уменьшит амплитуду цельноклеточная Са 2 + вспышек / переходных процессов. Сокращение семявыносящих протоков результатом сочетания в первую очередь purinergic и норадренергической нейротрансмиссии. Фокусное Са 2 + переходных процессов в этой ткани, однако, нечувствительны к норадренергической блокада рецепторов (например, α 1 адренорецепторов, по празозин 5), так что движение может быть уменьшена без изменения координационного Са 2 + переходных процессов. Кроме того, сокращение может быть предотвращено &quot;вниз&quot; через ингибирование миозина света, например, цепочку киназы вортманнином 6. Выводы Мониторинг Са 2 + флуоресценции при субмикронного разрешения мощную технику для изучения после соединительного эффекты нейромедиатора релиз 5,7 и выпуск Са 2 + из внутренних магазинах (например, «искры» 8). Анализ Са 2 + переходных использованием методологии, изложенной здесь является экономически эффективным по сравнению с коммерчески доступных пакетов анализа изображений и позволяет индивидуальные анализа с помощью специально написанный плагин Java для ImageJ.

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		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>Oregon Green 488 BAPTA-1 AM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>O6807</td>
<td>10 x 50 &mu;g</td>
</tr>
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<td>Pluronic F-127 solution</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>P3000MP</td>
<td>20% solution in DMSO</td>
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<td>Leica SP2 upright confocal microscope</td>
<td>&nbsp;</td>
<td>Leica</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Air table (&#x2018;vibration isolated workstation&#x2019;)</td>
<td>&nbsp;</td>
<td>Newport</td>
<td>M-VW-3046-OPT-012140</td>
<td>&nbsp;</td>
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<td>Sylgard 184 elastomer</td>
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focal ca2+ transient detection in smooth muscle

Са 2 + визуализации гладких мышц обеспечивает понимание клеточных механизмов, которые не могут привести к изменениям мембранного потенциала, таких, как освобождение Са 2 + из внутренних магазинах, а также позволяет несколько ячеек для мониторинга одновременно оценить, например, в связи синцитиальный ткани. Субклеточных Са 2 + переходных распространены в гладкой мускулатуре, но трудно точно измерить из-за проблем, вызванных их возникновения стохастических, более часто широкое поле зрения, в орган, который она склонна к контракту. Чтобы преодолеть эту проблему, мы разработали серию изображений протоколов и анализ процедур по приобретению и затем проанализировать, в автоматическом режиме, частоты, амплитуды и местоположение таких событий. Хотя такой подход может быть применен и в других контекстах, наша собственная работа включает в себя обнаружение локальных purinergic Са 2 + переходные для размещения передатчика релиз с резолюцией субмикронных. АТФ высвобождается в виде cotransmitter от вегетативных нервов, где он связывается с рецепторами на P2X1 гладкой мускулатуры детрузора и семявыносящих протоков. Са 2 + входит гладких мышц, в результате чего purinergic neuroeffector Са 2 + переходные (NCTS). Координационным Са 2 + переходных позволяют оптического мониторинга нейромедиатора выпуска таким образом, что имеет много преимуществ по сравнению с электрофизиологии. Помимо значительно улучшить пространственное разрешение, оптической записи имеет дополнительное преимущество, что позволяет запись передатчик освобождение от многих различимы сайтов одновременно. Кроме того, оптические плоскости фокуса легче поддерживать или исправить в течение длинного ряда записи, чем это репозиционирование внутриклеточных резкое микроэлектрода. Таким образом, метод для работы с изображениями Са 2 + флуоресценции в котором подробно изложил подготовки ткани, а также приобретение и анализ данных. Мы выделяем использованием нескольких сценариев для анализа таких Са 2 + переходных процессов.

Watch this Scientific Journal Video about Focal Ca2+ Transient Detection in Smooth Muscle at JoVE.com

Focal Ca2+ Transient Detection in Smooth Muscle

Подробнее методы высокого разрешения Ca<sup&gt; 2 +</sup&gt; Визуализации гладких мышц в изолированных органах, в том числе: подготовка ткани, получения изображений и анализа данных.

Фокусное Ca 2 + Переходные Обнаружение в гладкой мускулатуре

Ca2+ imaging of smooth muscle provides insight into cellular mechanisms that may not result in changes of membrane potential, such as the release of Ca2+ ...

focal-ca2-transient-detection-in-smooth-muscle

Research

JoVE Journal

Biology

Focal Ca2+ Transient Detection in Smooth Muscle

Isolation of Human Umbilical Arterial Smooth Muscle Cells (HUASMC)

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded and their collection does not imply any added risk to the newborn or mother s health. Moreover no ethical concerns are raised. The UC is composed by one vein and two arteries from which both endothelial cells (ECs) 1 and smooth muscle cells (SMCs) 2, two of the main cellular components of blood vessels, can be isolated. In this project the SMCs were obtained after enzymatic treatment of the UC arteries accordingly the experimental procedure previously described by Jaffe et al 3. After cell isolation they were kept in t-flash with DMEM-F12 supplemented with 5% of fetal bovine serum and were cultured for several passages. Cells maintained their morphological and other phenotypic characteristics in the different generations. The aim of this study was to isolate smooth muscle cells in order to use them as models for future assays with constrictor drugs, isolate and structurally characterize L-type calcium channels, to study cellular and molecular aspects of the vascular function 4 and to use them in tissue engineering.

Isolation of Human Umbilical Arterial Smooth Muscle Cells HUASMC

The umbilical cords are used to isolate smooth muscle cells by different ways. In this work we used the enzymatic treatment to isolated smooth muscle cells.

Изоляция по правам артерии пуповины гладкомышечных клеток (HUASMC)

The human umbilical cord (UC) is a biological sample that can be easily obtained just after birth. This biological sample is, most of the time, discarded ...

Exploring Arterial Smooth Muscle Kv7 Potassium Channel Function using Patch Clamp Electrophysiology and Pressure Myography

Contraction or relaxation of smooth muscle cells within the walls of resistance arteries determines the artery diameter and thereby controls flow of blood through the vessel and contributes to systemic blood pressure. The contraction process is regulated primarily by cytosolic calcium concentration ([Ca2+]cyt), which is in turn controlled by a variety of ion transporters and channels. Ion channels are common intermediates in signal transduction pathways activated by vasoactive hormones to effect vasoconstriction or vasodilation. And ion channels are often targeted by therapeutic agents either intentionally (e.g. calcium channel blockers used to induce vasodilation and lower blood pressure) or unintentionally (e.g. to induce unwanted cardiovascular side effects).
Kv7 (KCNQ) voltage-activated potassium channels have recently been implicated as important physiological and therapeutic targets for regulation of smooth muscle contraction. To elucidate the specific roles of Kv7 channels in both physiological signal transduction and in the actions of therapeutic agents, we need to study how their activity is modulated at the cellular level as well as evaluate their contribution in the context of the intact artery.
The rat mesenteric arteries provide a useful model system. The arteries can be easily dissected, cleaned of connective tissue, and used to prepare isolated arterial myocytes for patch clamp electrophysiology, or cannulated and pressurized for measurements of vasoconstrictor/vasodilator responses under relatively physiological conditions. Here we describe the methods used for both types of measurements and provide some examples of how the experimental design can be integrated to provide a clearer understanding of the roles of these ion channels in the regulation of vascular tone.

Measurements of Kv7 (KCNQ) potassium channel activity in isolated arterial myocytes (using patch clamp electrophysiological techniques) in parallel with measurements of constrictor/dilator responses (using pressure myography) can reveal important information about the roles of Kv7 channels in vascular smooth muscle physiology and pharmacology.

Изучение артериальной гладких мышц KV7 калиевых каналов функцию с помощью Patch Clamp электрофизиологии и давления Миография

Contraction  or relaxation of smooth muscle cells within the walls of resistance arteries  determines the artery diameter and thereby controls flow of ...

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 ...

Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method

Terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) is the method of using the TdT enzyme to covalently attach a tagged form of dUTP to 3&#8217; ends of double- and single-stranded DNA breaks in cells. It is a reliable and useful method to detect DNA damage and cell death in situ. This video describes dissection, tissue processing, sectioning, and fluorescence-based TUNEL labeling of mouse skeletal muscle. It also describes a method of semi-automated TUNEL signal quantitation. Inherent normal tissue features and tissue processing conditions affect the ability of the TdT enzyme to efficiently label DNA. Tissue processing may also add undesirable autofluorescence that will interfere with TUNEL signal detection. Therefore, it is important to empirically determine tissue processing and TUNEL labeling methods that will yield the optimal signal-to-noise ratio for subsequent quantitation. The fluorescence-based assay described here provides a way to exclude autofluorescent signal by digital channel subtraction. The TUNEL assay, used with appropriate tissue processing techniques and controls, is a relatively fast, reproducible, quantitative method for detecting apoptosis in tissue. It can be used to confirm DNA damage and apoptosis as pathological mechanisms, to identify affected cell types, and to assess the efficacy of therapeutic treatments in vivo.

Detection and Analysis of DNA Damage in Mouse Skeletal Muscle In Situ Using the TUNEL Method

This video describes dissection, tissue processing, sectioning, and fluorescence-based TUNEL labeling of mouse skeletal muscle. It also describes a method for semi-automated analysis of TUNEL labeling.

Выявление и анализ повреждений ДНК в клетках мышиной скелетных мышц<em&gt; На месте</em&gt; Использование Тюнеля метод

Terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) is the method of using the TdT enzyme to covalently ...

Measurement of Smooth Muscle Function in the Isolated Tissue Bath-applications to Pharmacology Research

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues. While this technique has been implemented for over 100 years, the versatility, simplicity and reproducibility of this assay helps it to remain an indispensable tool for pharmacologists and physiologists alike. Tissue bath systems are available in a wide array of shapes and sizes, allowing a scientist to evaluate samples as small as murine mesenteric arteries and as large as porcine ileum &#8211; if not larger. Central to the isolated tissue bath assay is the ability to measure concentration-dependent changes to isometric contraction, and how the efficacy and potency of contractile agonists can be manipulated by increasing concentrations of antagonists or inhibitors. Even though the general principles remain relatively similar, recent technological advances allow even more versatility to the tissue bath assay by incorporating computer-based data recording and analysis software. This video will demonstrate the function of the isolated tissue bath to measure the isometric contraction of an isolated smooth muscle (in this case rat thoracic aorta rings), and share the types of knowledge that can be created with this technique. Included are detailed descriptions of aortic tissue dissection and preparation, placement of aortic rings in the tissue bath and proper tissue equilibration prior to experimentation, tests of tissue viability, experimental design and implementation, and data quantitation. Aorta will be connected to isometric force transducers, the data from which will be captured using a commercially available analog-to-digital converter and bridge amplifier specifically designed for use in these experiments. The accompanying software to this system will be used to visualize the experiment and analyze captured data.

This protocol describes the measurement of isometric contraction in an isolated smooth muscle preparation, using an isolated tissue bath system and computer-based data acquisition.

Измерение гладких мышцах функции в выделенную ткань баня приложений к фармакологии исследований

Isolated tissue bath assays are a classical pharmacological tool for evaluating concentration-response relationships in a myriad of contractile tissues.  ...

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on changes in intracellular calcium ([Ca2+]i) and nitric oxide (NO). In order to understand how [Ca2+]i, NO and downstream molecules are handled by a blood vessel in response to vasoconstrictors and vasodilators, we developed a novel technique that applies calcium-labeling (or NO-labeling) dyes with two photon microscopy to measure calcium handling (or NO production) in isolated blood vessels. Described here is a detailed step-by-step procedure that demonstrates how to isolate an aorta from a rat, label calcium or NO within the endothelial or smooth muscle cells, and image calcium transients (or NO production) using a two photon microscope following physiological or pharmacological stimuli. The benefits of using the method are multi-fold: 1) it is possible to simultaneously measure calcium transients in both endothelial cells and smooth muscle cells in response to different stimuli; 2) it allows one to image endothelial cells and smooth muscle cells in their native setting; 3) this method is very sensitive to intracellular calcium or NO changes and generates high resolution images for precise measurements; and 4) described approach can be applied to the measurement of other molecules, such as reactive oxygen species. In summary, application of two photon laser emission microscopy to monitor calcium transients and NO production in the endothelial and smooth muscle cells of an isolated blood vessel has provided high quality quantitative data and promoted our understanding of the mechanisms regulating vascular function.

Two-photon Imaging of Intracellular Ca2+ Handling and Nitric Oxide Production in Endothelial and Smooth Muscle Cells of an Isolated Rat Aorta

Vascular cell functiondepends on activity of intracellular messengers. Described here is an ex vivo two photon imaging method that allows the measurement of intracellular calcium and nitric oxide levels in response to physiological and pharmacological stimuli in individual endothelial and smooth muscle cells of an isolated aorta.

Двухфотонное изображений внутриклеточного Ca<sup&gt; 2+</sup&gt; Погрузочно-разгрузочные работы и оксид азота производства в эндотелиальных и гладкомышечных клеток изолированной аорты крыс

Calcium is a very important regulator of many physiological processes in vascular tissues. Most endothelial and smooth muscle functions highly depend on ...

Isolation of Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

Выделение мышиной коронарных сосудов клетках гладкой мускулатуры

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

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.

Повреждение дрожь в аритмии: эффективный<em&gt; В Виво</em&gt; Модель гладкой мышечной клеточной пролиферации и эндотелиальной функции

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

Measuring Proliferation of Vascular Smooth Muscle Cells Using Click Chemistry

The ability of a cell to proliferate is integral to the normal function of most cells, and dysregulation of proliferation is at the heart of many disease processes. For these reasons, measuring proliferation is a common tool used to assess cell function. Cell proliferation can be measured simply by counting; however, this is an indirect means of measuring proliferation. One common means of directly detecting cells preparing to divide is by incorporation of labeled nucleoside analogs. These include the radioactive nucleoside analog 3H-thymidine plus non-radioactive nucleoside analogs such as 5-bromo-2&#8217; deoxyuridine (BrdU) and 5-ethynyl-2&#8242;-deoxyuridine (EdU). Incorporation of EdU is detected by click chemistry, which has several advantages when compared to BrdU. In this report, we provide a protocol for measuring proliferation by the incorporation of EdU. This protocol includes options for various readouts, along with the advantages and disadvantages of each. We also discuss places where the protocol can be optimized or altered to meet the specific needs of the experiment planned. Finally, we touch on the ways that this basic protocol can be modified for measuring other cell metabolites.

Proliferation is a critical part of cellular function, and a common readout used to assess potential toxicity of new drugs. Measuring proliferation is, therefore, a frequently used assay in cell biology. Here we present a simple, versatile method of measuring proliferation that can be used in adherent and non-adherent cells.

Измерение распространения сосудистых гладких мышечных клеток с помощью нажмите химии

The ability of a cell to proliferate is integral to the normal function of most cells, and dysregulation of proliferation is at the heart of many disease ...

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.

Выделение первичных клеток гладкой мускулатуры аорты, специфичных для пациента, и полуколичественные измерения сокращения в режиме реального времени 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 ...