Bu çalışma, Deutsche Forschungsgemeinschaft (KFO 196 &quot;Signaltransduktion bei adaptativen und maladaptiven kardialen Remodeling-Prozessen&quot;, 1638 / 1-2 FR) tarafından Kalp ve Damar Araştırma, Sağlık Araştırma Merkezleri Alman bir kısmı için DZHK (Almanca Merkezi tarafından desteklenen Bir BMBF (Eğitim ve Araştırma Alman Bakanlığı) girişimi) olan.

Not: izolasyon prosedürü temel adımları Şekil 1 &#39;de belirtilen etaplar aşağıdaki protokolde ayrıntılı olarak tarif edilmiştir.. Papiller kas izolasyonu, organ banyosu odasında montaj, toplama ve analiz ardışık ve zorunlu zaman ölçeğini yapılır. Tüm hayvan deneyleri hayvanların korunması Alman mevzuatına göre yapılmış ve Heidelberg Üniversitesi Etik Değerlendirme Kurulu tarafından onaylanmıştır. Enstrümantasyon hazırlanması 1. <ol><li> Kontraktilite için ölçümler multichambered organ banyosu kurulumu kullanın. Bir ex vivo kontraktilite organ banyosu kurulum bileşenleri, Şekil 2 de gösterilmiştir. NOT: papiller kas hazırlanması ve deney protokolünde başlangıcı arasındaki süreyi kurmak ekipman en aza indirmek ve deneysel tissue.This toplama provid için tamponlar önce hazırlamak içinDoku deneyler için canlı kalır zaman büyük miktarda es. </li><li> Bilgisayar ve veri toplama ekipmanı başlatın. Organ banyosu ısı (önceden ayarlanmış 32 ° C) açın ve önceden sıcaklığa birimleri izin verir. </li><li> Edinim ekipmanı hazırlayın. Yazılım kılavuzunda belirtildiği gibi verileri kaydetmeye başlayın. </li><li> Giriş ve her kanalda (gerekirse) kalibrasyonu yapmak ve sertifikalı ağırlıkça verilen bir 2-g kuvvetine (yani, kanal ile ilişkili) karşılıklı kuvvet dönüştürücü temin elektrik sinyali standardize etmek için tüm kanallar sıfır. </li><li> Organ banyosu odalarına gaz temini (% 95 O 2 /% 5 CO 2) açın. Sürekli tüm protokol ölçümleri sırasında tüm odaları gaz. </li></ol> Tamponlar ve Fizyolojik Çözümler 2. Hazırlık <ol><li> 119 mM NaCI nihai konsantrasyonlar elde etmek için Krebs-Henseleit tampon çözeltisi hazırlayın; 25 mM NaHCO 3; 4,6 mM KCI; 11mm glukoz; 1 mM Na-pyruvate; 1,5 mM CaCl2; 1.64 mM 4 MgSO; 1.18 mM KH 2 PO 4 ve (Tablo 1 Fizyolojik çözüm bakınız) 7.4 pH ayarlamak. </li><li> Ek 30 mM 2,3-Butandion monoxime (BDM) ve 50 IE Heparin / ml ile adım 2.1 &#39;de açıklandığı gibi izolasyonu sırasında kullanmak üzere ayrı Krebs-Henseleit tampon çözeltisi hazırlayın. 4 ° C (Tablo 1 Hazırlama çözeltisi bakınız) soğutun çözeltisi. Tablo 1 de tarif edildiği gibi işemi simülasyonu için, iskemi-solüsyon hazırlanır. </li><li> Ca 2 + protokolü için - bağımlı kontraktilite, istenen nihai konsantrasyona Krebs-Henseleit tampon çözeltisine Ca 2+ ekleyin. NOT: doğrudan kullanmadan önce bu çözümleri hazırlamak ve Calciumcarbonate çökmesini önlemek için karbojen ile Gass. Kullanmadan önce 32 ° C&#39;ye kadar ön ısıtılması çözeltisi. </li></ol> Papiller Eksizyon Prosedür sırasında kullanılan Gazlı Tüp ve Diseksiyon Dish 3. Hazırlık <ol><li> Yumuşak bir gaz bağlayınuzman çalışma ile gaz bağlantısı (% 100 oksijen, alternatif karbojen) tüp (dış çapı 2-4 mm) şarkı. </li><li> Diseksiyon çanak içine uydurma gazlama tüpün kapalı halka oluşturun. Gazlama tüpü sürekli köpürme güvence birkaç kez delinme. Bu gazlaştırma içi tüpü birkaç kez kullanılabilir. </li><li> Pimleri içine sıkışmış, böylece altta (0.5 cm kalınlığında) bir silikon elastomer diseksiyon çanak hazırlayın. Bu yemek birkaç kez kullanılabilir. </li></ol> Fareler ile Sol Ön Papiller kas 4. İzolasyonu NOT: papiller kas izolasyonu başlamadan önce, gaz hatları tıkanmalar açık olup olmadığını kontrol edin. <ol><li> Papiller kas hazırlık başlamadan önce protokol bölümünde 1-3 bahsedildiği gibi organ banyosu kurulumu hazırlayın. Deney günü tüm çözümleri hazırlayın. </li><li> Servikal dislokasyon accordi tarafından (yaklaşık 8-12 haftalık) fare Kurbanuzman çalışma ve kurumsal kurallarına ng. </li><li> Forepaws sabitleyerek dorsal pozisyonda hayvan düzeltmek ve bir diseksiyon gemide arka pençeleri, keskin kemik makasla kaburga ile keserek her iki tarafta toraks yanal açın, daha sonra bir çapraz kesim ile diyafram kesti. Perikard çıkarın. Şimdi kalp ve aort erişilebilir. </li><li> Kalbine yakın damar trunkustan üzerinde künt forseps ile kalbi düzeltmek ve akciğerlerden makas ve çevresindeki doku ile hızlı bir şekilde kalbi ayırın. </li><li> Oksijen (adım 2.2) ile gaz verilerek soğutulmuş hazırlık solüsyonu ile dolu bir Petri kabı kalpte aktarın. Kalp yendi ve forseps ile kardiyak apeks dokunmadan yoluyla usulca kalp kasılmalarını uyarmak için izin verin. </li><li> En kısa sürede kalbi tamamen kansızdırlar olduğunu O 2 ile soğutulan hazırlık solüsyonu (adım 2.2) ile doldurulur ve gazlı diseksiyon çanak aktarın. Kullanımı konusunda bu aşamada bir stereomikroskopta itibaren. </li><li> Hea FixKalp (sağ ventrikül operatörün görünümü sağ tarafta yer alan) dorsal görüldüğü şekilde sağ ventrikül ile küçük bir iğne ile rt. </li><li> Kullanımı mikrocerrahi makas ventriküllerin doku bağlayan atriyoventriküler düzeyde hem atriyumları kesmek ayırmak ve atın. Herhangi bir mekanik basınç kaçınarak kalbin apeks AV-valf-seviyesinden ventrikül duvarından bir kesim yapın. </li><li> Ventrikül açın ve dolayısıyla sol ventrikül papiller kas hem bakmak zorunda, forseps ile sol taraflı serbest duvarı sabitleyin. </li><li> Biraz papiller kas hazırlama kapak yelken bir kısmını koruyarak sol ön papiller kas her iki tarafında ventrikül doku yanal kesip ve dokunaklı önlemek veya mümkün olduğunca papiller kas germe. Papiller kas kalan ventrikül duvar dokusu teşrih. </li><li> Papiller kas hazırlık her iki tarafında takın ipek ipler (7/0, metrik 0.5)preparasyonu, kapak yelken biri ve kas parçası üzerinde tek. Sıcaklık 32 ° C olduğu ileri sürülmektedir, organ banyosu çözeltisi ile doldurulur ve karbojen gazı verilmiş organ banyosu odası içinde hazırlanmasını sabitleyin. </li></ol> Papiller kas 5. Dengeleme ve Uyarım <ol><li> Hemen organ banyosunda fiksasyon sonra 1 Hz stimülasyon frekansında dikdörtgen darbeler (2 ms süresi ve 100 mA akım) ile papiller kas hazırlık Canlandıracak. </li><li> Sürekli değişim ya da kullanılan organ banyosu Çeşidi kurulum göre sık manuel değişim (her 5 dk) yoluyla, organ banyosu çözeltisi sık değişiklik sağlayın. </li><li> Ulaştı giderek maksimal seğirme kuvveti kadar gösteriş artırın. Gösteriş artış seğirme yürürlükte bir başka güçlendirme tarafından takip değilken maksimal seğirme gücü ulaşılır. </li><li> Dengeleme 45-60 dakika sonra, deneysel protokol başlatın. </li></ol> NOT: Aşağıda belirtilen deneysel protokol fizyolojik ve patofizyolojik koşullarda kalp kasılmasını karakterize standart manevraları içermektedir. Temsili bölümde de temsili sonuçları gösteren ayrıntılı olarak bu protokolleri tanımlar (ayrıca bkz Tablo 2). <ol><li> Kayıt ve deneysel protokollerinin analizi <ol><li> Kayıt için yeterli temporal çözünürlük (satın alma oranı ≥ 1kHz) uygun bir yazılımı kullanın. </li><li> Seğirme kuvvet genliği (yükseklik), gerginlik (TTP) ve (örnek olarak bakınız Şekil 4, ADInstruments ve 5.5 Grafik) yazılım programında belirtildiği gibi yarım maksimal gevşeme zamanı (R 50 sırasıyla / TFall) zirve zaman gibi parametreleri analiz edin. </li></ol></li></ol>

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Yazarlar hiçbir rakip mali çıkarlarını var olduğunu beyan ederiz.

Bu yazıda farelerde kalp fizyolojisi ve patoloji ile ilişkili çok sayıda bilimsel sorulara cevap olarak transgenik hatların analizi ve yeni farmasötik yaklaşımların keşfedilmesi desteklemek için kullanılabilir in vitro fare papiller kas kontraktilitesini araştırmak için bir yöntem açıklanmaktadır Kalp bozuklukları tedavi etmek için. Biz kalp kası kasılma, fizyolojik, patolojik ve farmakolojik özelliklerini değerlendirmek için bu yöntemin kullanımını göstermektedir (Şekil ...

Kalp kasılmasında için rollerini atıfta iyon kanalları gibi proteinlerin incelenmesi, farklı patomekanizmaların keşfetmek ve iskemi ve kalp yetmezliği gibi kalp hastalıkları için yeni tedavi stratejilerinin oluşturulması esastır. Memeli kardiyomiyositlerde Kontraktil fonksiyonu çeşitli iyon kanalları, taşıyıcılar ve diğer proteinler ile modüle edilmesi bilinmektedir. Aksiyon potansiyeli 2+ kanalları dışı alandan Ca 2 + akını yol açar ve daha sonra Ca 2 + hücresel daralma 2. tetikler Ca 2 + salınımını (YÜÇAM) 1, -kaynaklı Ca voltaj bağımlı sarkolemmal L-tipi aktivasyonunu uyarılmış. Fizyolojik veya patolojik stres kalp kontraktilite ve adaptasyon merkezi bir rol oynar -signaling Ca 2 +. Katekolaminler böylece cAMP&#39;yi sentezler adenilil siklaz (AC) uyarıcı, kalp β-adrenerjik reseptörleri aktive. Aktif hale, protein,n Ca 2 + transientler ve kardiyak kontraktilite 1,3,4 modifikasyonu ile sonuçlanan L-tipi Ca 2 + kanalları, fosfolamban&#39;ın ve ryanodin reseptörleri gibi farklı hücre içi ve zar bağlı proteinleri fosforile A (PKA) kinaz. cAMP fosfodiesteraz (PDE) tarafından parçalanır. Β-adrenoseptörleri dışında Gs-bağlanmış reseptörlerin aktivasyonu, aynı zamanda, cAMP birikmesine yol açar. Izole ventriküler kas şeritlerinde kontraktilite ölçümleri tekniği de daha memeli türler 5-8 için kurulmuştur. Genin olasılığı farelerde hedef dayanarak fare kalp fizyolojisi analiz yöntemleri kurmak önemlidir. Ancak, farelerde izole kas hazırlıkları fizyolojik özellikleri hakkında varolan verileri deneysel koşullara 9-12 bağlı olarak değişiklik gösterir. Anlatılan yöntem sol ventrikül papiller kas öncesi kardiyak kontraktilite analiz etmek için kullanılırin vitro parations. Kardiyak kontraktilite incelenmesi kan basıncı, nörohümoral stimülasyonu ve fiziksel veya metabolik stres gibi in vivo olarak kardiyak kontraktiliteyi, modifiye etkilerin yokluğunda gerçekleştirilir. Müteahhitlik kas hazırlık dayak oranı titizlikle tanımlanmış ve keyfi değiştirilebilir. Seğirme kuvvet belirli kalsiyum konsantrasyonu gibi uyaranlara, frekans veya sıcaklık yenerek bağlamında analiz edilebilir. Buna ek olarak, bu yöntem, farklı sinyal geçiş yolu bileşenlerinin veya araştırmak ve yukarıda belirtilen deney koşullarının kontrol edilmesi suretiyle genetik olarak modifiye edilmiş fare modellerinde kalp performansını karşılaştırmak için kullanılabilir.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S5761&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>P9333</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>D9434&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Sigma-Aldrich</td>
			<td>P5280&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate</td>
			<td>Sigma-Aldrich</td>
			<td align="right">223506</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate heptahydrate</td>
			<td>Sigma-Aldrich</td>
			<td align="right">230391</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Sigma-Aldrich</td>
			<td>P 5655</td>
			<td></td>
		</tr>
		<tr>
			<td>2,3-Butanedione monoxime</td>
			<td>Sigma-Aldrich</td>
			<td>B0753</td>
			<td></td>
		</tr>
		<tr>
			<td>Forskolin</td>
			<td>Sigma-Aldrich</td>
			<td>F3917</td>
			<td>Hazard statement H312, solve in DMSO</td>
		</tr>
		<tr>
			<td>3-?Isobutyl-?1-?methylxanthine</td>
			<td>Sigma-Aldrich</td>
			<td>I5879</td>
			<td>Hazard statement H 302, solve in DMSO</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoprenaline hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>I5627</td>
			<td>Hazard statement H 315-H319-H335</td>
		</tr>
		<tr>
			<td>Sodium Heparine 250.000 IE/10ml</td>
			<td>ratiopharm</td>
			<td>PZN 3874685</td>
			<td></td>
		</tr>
		<tr>
			<td>Histamine dihydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>H7250</td>
			<td>Hazard statement H 315-H 317-H319- H334-H335</td>
		</tr>
	</tbody>
</table>

contractility measurements on isolated papillary muscles for the investigation of cardiac inotropy in mice

Yetişkin fare kalpleri izole papiller kas farklı fizyolojik / patolojik koşullar kardiyak kontraktiliteyi incelemek için kullanılabilir. Kasılma özellikleri bağımsız gibi damar tonusunu veya nörohumoral durumu gibi dış etkilerden değerlendirilebilir. Bu izole kardiyak miyositler tek hücreli ölçümler arasında ve ekokardiyografi gibi in vivo çalışmalarda bilimsel bir yaklaşım gösteriyor. Mükemmel bir model kalp fizyolojisi / patofizyolojisini ve farmakolojik maddeler ile modülasyon ve transgenik hayvan modelleri keşfi gibi araştırmalar için kullanılabilir Dolayısıyla, papiller kas preparasyonlar vermektedir. Burada, bir organ banyosu kurulumunda kardiyak kontraktilite araştırmak için fare sol ön papiller kas izole bir yöntem açıklanmaktadır. Ventriküler duvara izole edilmiş bir kas şerit hazırlanmasında aksine, papiller kas, kas tissu zarar vermeden toto hazırlanabilirciddi e. Organ banyosu kurulumun birkaç ısı kontrollü, gazlanmış ve elektrot donatılmış organ banyosu daireden oluşur. İzole papiller kas organ banyosu odasında sabit ve elektriksel uyarılır. Uyarılmış seğirme gücü gibi kuvvet genliği seğirme ve kinetik analiz edilir seğirme gibi basınç dönüştürücü ve parametreler kullanılarak kaydedilir. Farklı deney protokolleri, katekolaminlerin ya da diğer ilaç olarak kontraktil ajanlar, kalsiyum ve frekansa bağlı kasılma yanı sıra doz-tepki eğrileri araştırmak için gerçekleştirilebilir. Ayrıca, akut iskemi gibi patolojik durumlar simüle edilebilir.

İzole fare papiller kas hazırlıkları kontraktilite ölçümleri için bu yazının protokol fizyolojik koşullar altında tekrarlanabilir deneysel sonuçlar elde etmek için optimum koşullara ayarlanmıştır. Optimal deneysel koşullar tanımlamak için biz organ banyosu sıcaklığı ve hücre dışı kalsiyum konsantrasyonu (ayrıca 12 bakınız) değişen Pilot deneyleri yaptık. Burada açıklanan protokol 1,5 mM hücre dışı kalsiyum konsantrasyonu ile 32 ° C arasındaki bir sıcaklıkta gerçek...

Watch this Scientific Journal Video about Contractility Measurements on Isolated Papillary Muscles for the Investigation of Cardiac Inotropy in Mice at JoVE.com

Contractility Measurements on Isolated Papillary Muscles for the Investigation of Cardiac Inotropy in Mice

Murin sol ventrikül papiller kas in vitro kardiyak kontraktiliteyi araştırmak için kullanılabilir. Bu makalede ayrıntılı olarak izolasyon ve deneysel protokoller kalp kasılma özelliklerini incelemek için açıklar.

Farelerde Kardiyak inotropiyi Soruşturma İzole papiller Kaslar kontraktilite Ölçümleri

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The ...

contractility-measurements-on-isolated-papillary-muscles-for

Research

JoVE Journal

Biology

14.9K Görüntüleme.  Universität Heidelberg.  Murin sol ventrikül papiller kas in vitro kardiyak kontraktiliteyi araştırmak için kullanılabilir. Bu makalede ayrıntılı olarak izolasyon ve deneysel protokoller kalp kasılma özelliklerini incelemek için açıklar. 

Video: Contractility Measurements on Isolated Papillary Muscles for the Investigation of Cardiac Inotropy in Mice

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

İyon Kanalları incelenmesi için yama Kelepçe ve Perfüzyon Teknikleri cinsinden ifade edilir Xenopus Oosit

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Biyoloji

Bioengineering Human Microvascular Networks in Immunodeficient Mice

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks in vivo. In this regard, the search for experimental models to build blood vessel networks in vivo is of utmost importance 1. The feasibility of bioengineering microvascular networks in vivo was first shown using human tissue-derived mature endothelial cells (ECs) 2-4; however, such autologous endothelial cells present problems for wide clinical use, because they are difficult to obtain in sufficient quantities and require harvesting from existing vasculature. These limitations have instigated the search for other sources of ECs. The identification of endothelial colony-forming cells (ECFCs) in blood presented an opportunity to non-invasively obtain ECs 5-7. We and other authors have shown that adult and cord blood-derived ECFCs have the capacity to form functional vascular networks in vivo 7-11. Importantly, these studies have also shown that to obtain stable and durable vascular networks, ECFCs require co-implantation with perivascular cells. The assay we describe here illustrates this concept: we show how human cord blood-derived ECFCs can be combined with bone marrow-derived mesenchymal stem cells (MSCs) as a single cell suspension in a collagen/fibronectin/fibrinogen gel to form a functional human vascular network within 7 days after implantation into an immunodeficient mouse. The presence of human ECFC-lined lumens containing host erythrocytes can be seen throughout the implants indicating not only the formation (de novo) of a vascular network, but also the development of functional anastomoses with the host circulatory system. This murine model of bioengineered human vascular network is ideally suited for studies on the cellular and molecular mechanisms of human vascular network formation and for the development of strategies to vascularize engineered tissues.

Here, we describe a methodology to deliver human cord blood-derived endothelial colony-forming cells (ECFCs) and bone marrow-derived mesenchymal stem cells (MSCs), embedded in a collagen/fibronectin gel, subcutaneously into immunodeficient mice. This cell/gel combination generates a human vascular network that connects with the mouse vasculature.

Bağışık yetmezliği olan farelere Biyomühendislik İnsan Mikrovasküler Ağları

The future of tissue engineering and cell-based therapies for tissue regeneration will likely rely on our ability to generate functional vascular networks ...

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

The isolated microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of factors that control vessel diameter, and thus, perfusion resistance1-5. This is a classic experimental preparation that was, in large measure, initially described by Uchida et al.15 several decades ago. This initial description provided the basis for the techniques that was extensively modified and enhanced, primarily in the laboratory of Dr. Brian Duling at the University of Virginia6-8, and we present a current approach in the following pages. This preparation will specifically refer to the gracilis arteriole in a rat as the microvessel of choice, but the basic preparation can readily be applied to vessels isolated from nearly any other tissue or organ across species9-13. Mechanical (i.e., dimensional) changes in the isolated microvessels can easily be evaluated in response to a broad array of physiological (e.g., hypoxia, intravascular pressure, or shear) or pharmacological challenges, and can provide insight into mechanistic elements comprising integrated responses in an intact, although ex vivo, tissue. The significance of this method is that it allows for facile manipulation of the influences on the integrated regulation of microvessel diameter, while also allowing for the control of many of the contributions from other sources, including intravascular pressure (myogenic), autonomic innervation, hemodynamic (e.g., shear stress), endothelial dependent or independent stimuli, hormonal, and parenchymal influences, to provide a partial list. Under appropriate experimental conditions and with appropriate goals, this can serve as an advantage over in vivo or in situ tissue/organ preparations, which do not readily allow for the facile control of broader systemic variables. 
The major limitation of this preparation is essentially the consequence of its strengths. By definition, the behavior of these vessels is being studied under conditions where many of the most significant contributors to the regulation of vascular resistance have been removed, including neural, humoral, metabolic, etc. As such, the investigator is cautioned to avoid over-interpretation and extrapolation of the data that are collected utilizing this preparation. The other significant area of concern with regard to this preparation is that it can be very easy to damage cellular components such as the endothelial lining or the vascular smooth muscle, such that variable source of error can be introduced. It is strongly recommended that the individual investigator utilize appropriate measurements to ensure the quality of the preparation, both at the initiation of the experiment and periodically throughout the course of a protocol.

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

An ex vivo preparation is described for isolation of the largest gracilis muscle resistance arterioles for interrogation of both vascular responses to vasoactive stimuli and the assessment of basic structural properties via passive wall mechanics.

Ex vivo</emVasküler Reaktivite incelenmesi için> İzole İskelet mikrodamar Hazırlık

The isolated  microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of  factors that control vessel ...

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, fatigability, and recovery after fatigue can be obtained to assess specific aspects of the excitation-contraction coupling (ECC) process such as excitability, contractile machinery and Ca2+ handling ability. This method removes the nerve and blood supply and focuses on the isolated skeletal muscle itself. We routinely use this method to identify genetic components that alter the contractile property of skeletal muscle though modulating Ca2+ signaling pathways. Here, we describe a newly identified skeletal muscle phenotype, i.e., mechanic alternans, as an example of the various and rich information that can be obtained using the in vitro muscle contractility assay. Combination of this assay with single cell assays, genetic approaches and biochemistry assays can provide important insights into the mechanisms of ECC in skeletal muscle.

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

We describe a method to directly measure muscle force, muscle power, contractile kinetics and fatigability of isolated skeletal muscles in an in vitro system using field stimulation. Valuable information on Ca2+ handling properties and contractile machinery of the muscle can be obtained using different stimulating protocols.

İzole İskelet Kaslar Kasılma, çabuk yorulma ve Alternans Ex Vivo Değerlendirilmesi

 
Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, ...

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to nonmarrow tissue have not been completely clarified. We describe here the technique of intravital microscopy applied to the mouse cremaster microcirculation for analysis of peripheral bone marrow stem cell migration in vivo. Intravital microscopy of the M. cremaster has been previously introduced in the field of inflammatory research for direct observation of leucocyte interaction with the vascular endothelium. Since sufficient peripheral stem and progenitor cell migration includes similar initial steps of rolling along and firm adhesion at the endothelial lining it is conceivable to apply the M. cremaster model for the observation and quantification of the interaction of intravasculary administered stem cells with the endothelium. As various chemical components can be selectively applied to the target tissue by simple superfusion techniques, it is possible to establish essential microenvironmental preconditions, for initial stem cell recruitment to take place in a living organism outside the bone marrow.

Intravital microscopy of the mouse M. cremaster microcirculation offers a unique and well-standardized in vivo model for the analysis of peripheral bone marrow stem cell migration.

Periferik Kök Hücre Göç Analizi Fare Cremaster Muscle Mikrosirkülasyon Intravital Mikroskopi

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to ...

The Mouse Isolated Perfused Kidney Technique

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a prerequisite for studying the physiology of the isolated organ and for many innovative applications that may be possible in the future, including perfusion decellularization for kidney bioengineering or the administration of anti-rejection or genome-editing drugs in high doses to prime the kidney for transplantation. During the time of the perfusion, the kidney can be manipulated, renal function can be assessed, and various pharmaceuticals administered. After the procedure, the kidney can be transplanted or processed for molecular biology, biochemical analysis, or microscopy.
This paper describes the perfusate and the surgical technique needed for the ex vivo perfusion of mouse kidneys. Details of the perfusion apparatus are given and data are presented showing the viability of the kidney&#39;s preparation: renal blood flow, vascular resistance, and urine data as functional, transmission electron micrographs of different nephron segments as morphological readouts, and western blots of transport proteins of different nephron segments as molecular readout.

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. The buffers and surgical technique are described in detail.

Fare İzole perfüze Böbrek Tekniği

The mouse isolated perfused kidney (MIPK) is a technique for keeping a mouse kidney under ex vivo conditions perfused and functional for 1 hr. This is a ...

Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents in the treatment of several diseases. The development of new, innovative strategies for miRNA/siRNA therapy is based on an extensive knowledge of the underlying mechanisms. Recent data suggest that small RNAs are exchanged between cells in a gap junction-dependent manner, thereby inducing gene regulatory effects in the recipient cell. Molecular biological techniques and flow cytometric analysis are commonly used to study the intercellular exchange of miRNA. However, these methods do not provide high temporal resolution, which is necessary when studying the gap junctional flux of molecules. Therefore, to investigate the impact of miRNA/siRNA as intercellular signaling molecules, novel tools are needed that will allow for the analysis of these small RNAs at the cellular level. The present protocol describes the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) microscopy to elucidating the gap junction-dependent exchange of miRNA molecules between cardiac cells. Importantly, this straightforward and non-invasive live-cell imaging approach allows for the visualization and quantification of the gap junctional shuttling of fluorescently labeled small RNAs in real time, with high spatio-temporal resolution. The data obtained by 3D-FRAP confirm a novel pathway of intercellular gene regulation, where small RNAs act as signaling molecules within the intercellular network.

Here, we describe the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) for the analysis of the gap junction-dependent shuttling of miRNA. In contrast to commonly applied methods, 3D-FRAP allows for the quantification of the intercellular transfer of small RNAs in real time, with high spatio-temporal resolution.

3D-FRAP Mikroskopisi ile Gap Birleşimine Bağlı MiRNA Transferinin Analizi

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents ...

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Gene manipulation specifically in the heart significantly potentiate the investigation of cardiac disease pathomechanisms and their therapeutic potential. In vivo cardiac-specific gene delivery is commonly achieved by either systemic or local delivery. Systemic injection via tail vein is easy and efficient in manipulating cardiac gene expression by using recombinant adeno-associated virus 9 (AAV9). However, this method requires a relatively high amount of vector for efficient transduction, and may result in nontarget organ gene transduction. Here, we describe a simple, efficient, and time-saving method of intramyocardial injection for in vivo cardiac-specific gene manipulation in mice. Under anesthesia (without ventilation), the pectoral major and minor muscles were bluntly dissected, and the mouse heart was quickly exposed by manual externalization through a small incision at the fourth intercostal space. Subsequently, adenovirus encoding luciferase (Luc) and vitamin D receptor (VDR), or short hairpin RNA (shRNA) targeting VDR, was injected with a Hamilton syringe into the myocardium. Subsequent in vivo imaging demonstrated that luciferase was successfully overexpressed specifically in the heart. Moreover, Western blot analysis confirmed the successful overexpression or silencing of VDR in the mouse heart. Once mastered, this technique can be used for gene manipulation, as well as injection of cells or other materials such as nanogels in the mouse heart.

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Here we present a protocol for cardiac-specific gene manipulation in mice. Under anesthesia, the mouse hearts were externalized through the fourth intercostal space. Subsequently, adenoviruses encoding specific genes were injected with a syringe into the myocardium, followed by protein expression measurement via in vivo imaging and Western blot analysis.

Vivo içinde kalp özel gen manipülasyon farelerde Intramyocardial enjeksiyonla için basit ve etkili bir yöntem

Gene manipulation specifically in the heart significantly potentiate the investigation of cardiac disease pathomechanisms and their therapeutic potential. ...

Analyzing the &alpha;-Actinin Network in Human iPSC-Derived Cardiomyocytes Using Single Molecule Localization Microscopy

The maturation of iPSC-derived cardiomyocytes is a critical issue for their application in regenerative therapy, drug testing and disease modeling. Despite the development of multiple differentiation protocols, the generation of iPSC cardiomyocytes resembling an adult-like phenotype remains challenging. One major aspect of cardiomyocytes maturation involves the formation of a well-organized sarcomere network to ensure high contraction capacity. Here, we present a super resolution-based approach for semi-quantitative analysis of the &#945;-actinin network in cardiomyocytes. Using photoactivated localization microscopy a comparison of sarcomere length and z-disc thickness of iPSC-derived cardiomyocytes and cardiac cells isolated from neonatal tissue was performed. At the same time, we demonstrate the importance of proper imaging conditions to obtain reliable data. Our results show that this method is suitable to quantitatively monitor the structural maturity of cardiac cells with high spatial resolution, enabling the detection of even subtle changes of sarcomere organization.

Analyzing the &#945;-Actinin Network in Human iPSC-Derived Cardiomyocytes Using Single Molecule Localization Microscopy

The formation of a proper sarcomere network is important for the maturation of iPSC-derived cardiomyocytes. We present a super resolution-based approach that allows for the quantitative evaluation of the structural maturation of stem cell derived cardiomyocytes, to improve culture conditions promoting cardiac development.

Tek Moleküllü Lokalizasyon Mikroskobu Kullanılarak İnsan iPSC Kaynaklı Kardiyomiyositlerde α-Aktinin Ağının Analizi

The maturation of iPSC-derived cardiomyocytes is a critical issue for their application in regenerative therapy, drug testing and disease modeling. ...

Dissection of the Endolymphatic Sac from Mice

The study of mutant mouse models of human hearing and balance disorders has unraveled many structural and functional changes which may contribute to the human phenotypes. Although important progress has been done in the understanding of the development and function of the neurosensory epithelia of the cochlea and vestibula, limited knowledge is available regarding the development, cellular composition, molecular pathways and functional characteristics of the endolymphatic sac. This is, in large part, due to the difficulty of visualizing and microdissecting this tissue, which is an epithelium comprised of only one cell layer. The study presented here describes an approach to access and microdissect the endolymphatic sac from the wild-type mouse inner ear at different ages. The result of a similar dissection is shown in a pendrin-deficient mouse model of enlargement of the vestibular aqueduct. A transgenic mouse with a fluorescent endolymphatic sac is presented. This reporter mouse can be used to readily visualize the endolymphatic sac with limited dissection and determine its size. It can also be used as an educational tool to teach how to dissect the endolymphatic sac. These dissection procedures should facilitate further characterization of this understudied part of the inner ear.

<meta charset="utf8"></head><body>Farelerden Endolenfatik Kesenin Diseksiyonu

This study describes how to dissect the endolymphatic sac from the inner ear of the mouse at different ages. The result of a similar dissection is shown in an Slc26a4-null mouse model of enlargement of the endolymphatic sac. A transgenic mouse with a fluorescent reporter expressed in the endolymphatic sac is presented as a model to readily visualize the endolymphatic sac, and educational tool.

Farelerden Endolenfatik Kesenin Diseksiyonu

The study of mutant mouse models of human hearing and balance disorders has unraveled many structural and functional changes which may contribute to the ...

Farelerde Kardiyak inotropiyi Soruşturma İzole papiller Kaslar kontraktilite Ölçümleri

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