Çalıştaylar ve Nörofizyoloji Enstitüsünün hayvan tesisi tarafından sağlanan desteği onaylıyoruz. Bu çalışma Walter und Marga Boll Stiftung, Köln Fortune ve Deutsche Stiftung für Herzforschung tarafından desteklendi.

Hayvanların taşınması, yerel hayvan refahı komitesinin ve Avrupa Parlamentosu&#39;nun 2010/63 / AB sayılı Direktifinin kurallarına uymalıdır. 1. Çözümleri Hazırlama <ol><li> Tyrode&#39;nin solüsyonunu Ca 2+ olmadan (mM&#39;de kompozisyon) hazırlayın: NaCI 136, KCl 5.4, NaH2PO4.33, MgCl2, glikoz 10, HEPES 5, 2,3-butandion monoksim (BDM) 30. pH&#39;ı 7.4&#39;e ayarlayın NaOH ile 4 ° C&#39;de. </li><li> Tyrode çözeltisini Ca 2+ (bileşim mM cinsinden) ile hazırlayın: NaCl 136, KCl 5.4, NaH2PO4.33, MgCl2, glikoz 10, HEPES 5, BDM 30, CaCl 2 0.9. 4 ° C&#39;de NaOH ile pH&#39;ı 7.4&#39;e ayarlayın. </li><li> % 4 düşük eriyikli agaroz hazırlayın: Ca 2+ içermeyen 0.6 mL düşük eriyikli agaroz 15 mL Tyrode çözeltisine koyun. Karışımı, bir mikrodalga fırında iki kez 750 W&#39;de 10-15 saniye boyunca, agaroz çözünene kadar ısıtın. Çözeltiyi 37 ° C&#39;de sabit bir sıcaklıkta tutun.# 176; C ve sürekli karıştırın. </li><li> Dulbecco&#39;nun Değiştirilmiş Kartal Ortamını (DMEM) 37 ° C&#39;de serum olmadan carbogen (% 5 CO 2 ,% 95 O 2 ) ile kabarcıklarken muhafaza edin. </li></ol> 2. Microtome&#39;u hazırlayın <ol><li> Mikrogramı açın. </li><li> Dış mikrotom hücresini buzla doldurun. </li><li> İç mikrotom bölmesini, Ca 2+ içermeyen soğuk Tyrode çözeltisi ile doldurun ve çözeltiyi% 100 O 2 ile sürekli olarak oksijene koyun. </li><li> Mikro topuzun bıçak tutucusuna bir çelik bıçak takın. </li></ol> 3. Fare Kalp İzolasyonu <ol><li> 2,500 U Heparin&#39;i subkütan enjekte edin. 15 dakika bekleyin. </li><li> Hayvanı servikal dislokasyon ile kurban edin. </li><li> Sternotomi ile göğsünü açın. </li><li> Küçük makas ve bir forseps # 5 kullanarak perikardı dikkatle inceleyin. </li><li> Artan aortaya bir kanül yerleştirin ve koroner arterleri buz gibi soğuk Tyrode&#39;lerle in situ olarak perfüze edinKalan kan alınana kadar Ca 2+ içermeyen bir solüsyon. </li><li> Kalbi bir forseps ve makasla hafifçe kesip kalbi buz soğukluğunda transfer edin Tyrode&#39;nin çözümü Ca 2+ olmadan sürdü. </li><li> Atriyumu ventriküllerden bir neşter veya makasla ayırın. </li></ol> 4. Ventriküllerin% 4 Düşük Erime Agaroza Katılması <ol><li> Ventrikülleri, tepe kısmı yukarı bakacak şekilde agaroz kalıbına yerleştirin ( Şekil 1 ). Pimi sol ventriküler bölmedeki kalıbın ortasına yerleştirin. </li><li> Kalıbı, kalp tamamen kaplanıncaya kadar 37 ° C&#39;de% 4 düşük eriyikli agaroz ile doldurun. </li><li> Agarozun daha hızlı sertleşmesi için kalıbı buz üzerinde bırakın, dokunun yüzmesini önleyin. </li><li> Ventrikül içeren agaroz bloğu bir bistül ile kalıptan çıkarın. </li><li> Bloğu ters çevirin ve ventriküler odaları ve agaroz bloğun arkasındaki boşluğu doldurun;20 G&#39;lik bir iğneli bir şırınga kullanarak% 4 düşük eriyikli agaroz ile moltun iğnesi bıraktı. </li><li> Blok düz bir alt ve kalp apeksinin dik pozisyonunu elde etmek için agaroz bloğu bir neşter ile kesin. </li></ol> 5. Ventriküler Doku Dilimleme <ol><li> Bir damla cyanoacrylate tutkalı ile mikrotomun numune tutucusundaki bloğu sabitleyin. Kardiyak üstü yukarı bakacak şekilde tasarlayın. </li><li> Örnek tutucuyu, Ca 2+ içermeyen buz gibi soğuk Tyrode ile dolu mikrotomun iç numune bölmesine yerleştirin. Tyrode&#39;nin çözeltisiyle agaroz bloğu tamamen örtün. </li><li> Diğer uygulamalara (keskin elektrot kayıtları 150-200 μm için) bağlı olarak 150-400 μm kalınlığında kısa eksen dilimleri hazırlayın, bıçağın titreşim frekansı 60-70 Hz&#39;de tutun ve bıçağı mümkün olduğunca yavaş ileri hareket ettirin . </li><li> Kalan agarozu dilimlerden dikkatlice çıkarmak için ince bir fırça kullanın. </li><li> Nazikçe gönderR dilimleri,% 0.9 O 2 ile havalandırılan 0.9 mM Ca 2 + ile Tyrode solüsyonuna bir Pasteur pipet ile dilimleme prosedüründen kurtulmak için buz üzerinde en az 30 dakika saklayın. </li><li> Ardından, daha fazla kullanılmadan önce BDM&#39;yi yıkamak için 30 dakika süreyle DMEM&#39;de 37 ° C&#39;de dilimler bırakın, karbogenle havalandırın. </li></ol> 6. Keskin Elektrot Kurulumunun Hazırlanması <ol><li> Ön ısıtma için, kayıtların başlamasından 30 dakika önce tüm elektrikli cihazları açın. </li><li> Ters çevrilmiş mikroskopta yer alan ısıtma plakasına 3 cm&#39;lik hücre kültürü kabı koyun. </li><li> Özel yapım halka elektrotunu ( Şekil 2 ) çanağa yerleştirin ve ön amplifikatörün ve stimülasyon elektrodunun topraklama kablolarını bağlayın. </li><li> Perfüzyon sisteminin esnek borularını çanağa bağlayın. </li><li> Perfüzyon sisteminin rezervuarını karbogen ile havalandırılmış DMEM ile doldurun. </li><li> Perfüzyon pompasını açın ve perfüzyon rafini ayarlayın2-3 mL / dk&#39;ya kadar ısıtıldı. </li><li> Akış ısıtıcısını ve ısıtma plakasını düzenleyerek çanak içindeki DMEM sıcaklığını 37 ° C&#39;ye ayarlayın. </li></ol> 7. İşlem Potansiyel Kayıtları <ol><li> DMEM dolu tabağa bir ventriküler dilim yerleştirin. </li><li> Ters çevrilmiş mikroskop ile yapının yapısal bütünlüğünü ve yaşayabilirliğini kontrol edin (kontraktil fonksiyona dayanmaktadır). </li><li> Bir kayıt cam elektrodu 3 M KCL ile doldurun. </li><li> Bir uyarılma elektrotunu DMEM ile doldurun. </li><li> Kayıt elektrodunu ve stimülasyon elektrodunu elektrot tutucularının üzerine yerleştirin. </li><li> Uyarı elektrodunu dilim üzerine dikkatle yerleştirin ve elektrik stimülatörünü açın. 1-2 Hz&#39;lik bir stimülasyon frekansı ile başlayın. </li><li> Kayıt elektrodunu mikromanipülatör ile istenen kayıt pozisyonunun üzerine getirin. </li><li> İpucu dokuya dokunana kadar kayıt elektrodunu yavaşça indirin. </li><li> Şebekeden kısa dikdörtgen elektrik darbesi uygulayın.Kayıt elektrotu hücre zarı içine nüfuz eder. </li><li> Kayıt elektrodunu kararlı bir sinyal gelene kadar dikkatlice yeniden konumlandırın. </li><li> Aksiyon potansiyellerinin kaydını başlatın. </li></ol>

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Yazarların beyan edecek hiçbir şeyi yok.

Ventriküler dilimler, korunmuş bir in vivo doku yapısı ve ölçme teknolojisinin kalbin tüm bölgelerine doğrudan erişimi ile elektrofizyolojik, farmakolojik ve mekanik çalışmalara olanak tanır. Fizyolojik aksiyon potansiyel özellikleri embriyonik, neonatal ve erişkin dilimlerde gösterilmiştir 9,10,11. Dilimleme işlemi ile doğrudan hasar gören yüzey tabakaları haricindeki dilimlerin canlılığı, canlılık boyaması ile teyit edilmiştir 9 . Burada açıklandığı ...

Yamamot ve Mcllwain 1966&#39;da beyin dilimlerinin elektriksel aktivitesinin in vitro muhafaza edildiğini gösterdiklerinden beri temel bilimde ince doku dilimleri sıklıkla kullanılmıştır. O zamandan beri, beyin 2 , karaciğer 3 , akciğer 4 ve miyokard dokusu 5 , 6 , 7 dilimleri üzerinde elektrofizyolojik ve farmakolojik çalışmalar yürütülmüştür. Yenidoğan sıçan kalplerindeki ventriküler dilimlerde ilk yama-kelepçe kayıtları 1990&#39;da 8&#39;de tanımlanmıştır, ancak bu teknik bir süre unutulmaya uğradı. Birkaç yıl sonra grubumuz, fare embriyonik 9 , neonatal 10 ve erişkin 11 kalp dilimlerini hazırlamak için yeni bir yöntem geliştirdi. Bu canlı doku dilimleri akut deneyler için kullanılabilir (erişkin dilimS birkaç saat ekilebilir) veya kısa vadeli kültür deneyleri (embriyonik ve yenidoğan dilimleri birkaç gün boyunca yetiştirilebilir). Kesitler elektrofizyolojik özelliklere benzer şekilde in vivo olarak gösterirler ve keskin elektrot aksiyon potansiyeli ve mikro elektrot dizisi kayıtları ile değerlendirildiğinde homojen bir uyarılma yayılır 11 . &quot;İki boyutlu&quot; morfolojilerinden dolayı kayıt elektrotlarının ventrikülün tüm bölgelerine doğrudan erişmelerine izin veriyorlar, bu da elektrofizyolojik araştırmalar için ilginç bir araç haline getiriyor ve Langendorff tarafından perfüze edilmiş kalpler ile karşılaştırıldığında yeni deneysel seçenekler getiriyor. Dilimlerin verapamil (L-tipi Ca2 + -kanal bloke edicisi), lidokain (Na + -kanal bloke edicisi), 4-aminopiridin (seçici olmayan voltaja bağımlı K + -kanal bloke edici) ve linopirdin (KCNQ K) gibi ilaçların iyon tutucu etkisi + -kanal bloke edici) 9 , 11 </suP&gt; ayrışmış kardiyomiyositlerde bilinen etkilere karşılık gelmektedir. İzometrik kuvvet ölçümleri pozitif bir kuvvet frekansı ilişkisi ortaya koydu ve güçlü kasılma fonksiyonu önerdi10. Bu bulgular, sıçanın ventriküler dilimlerinin fizyolojik ve farmakolojik çalışmalar için in vitro bir doku modeli olarak uygun olduğunu göstermiştir. Ayrıca, kesici elektrot kayıtları ile birlikte alıcı kalplerin ventriküler dilimleri, transplanted fetal 12 , 13 , 14 ve kök hücre kaynaklı 15 kardiyomiyositlerin olgunlaşması yanı sıra elektriksel ve mekanik entegrasyonu karakterize etmek için çok yararlı bir araç olduğu kanıtlanmıştır. Özetle, ventriküler dilimler değerli ve iyi kurulmuş bir çok hücreli doku modeli olup ayrışmış kardiyomiyositleri ve Langendorff perfüze edilen kalpler için tamamlayıcı olarak düşünülmelidir(Dissociated hücrelere karşıt olarak) bir in vivo benzeri doku yapısı sağlamanın yanı sıra keskin elektrot kayıtları gibi (kalp preparatlarının aksine) kalbin tüm bölgelerine doğrudan erişim sağlayan en büyük avantajı olan kardiyovasküler araştırmalardır.

<table><tbody><tr><td>Leica VT 1000s</td><td>Leica Microsystems, Wetzlar, Germany</td><td></td><td>Microtome with vibrating blade.</td></tr><tr><td>Stainless Steel Blades</td><td>Campden Instruments, Loughborough, England</td><td>7550-1-SS</td><td></td></tr><tr><td>Pasteur pipettes</td><td>Sigma-Aldrich, St. Louise, USA</td><td>Z627992</td><td></td></tr><tr><td>Fine brush, &lt;em&gt;e.g.&lt;/em&gt; size 6 (4/32&quot;)</td><td>VWR, International, Radnor, USA</td><td>149-2125</td><td></td></tr><tr><td>Preparation table</td><td></td><td></td><td>self made</td></tr><tr><td>Molt for embedding ventricles in agarose</td><td></td><td></td><td>self made</td></tr><tr><td>1 mL Syringe</td><td>Becton, Dickinson; Franklin Lakes, USA</td><td>300013</td><td></td></tr><tr><td>27 G x 3/4`` Needles</td><td>Braun, Melsungen, Germany</td><td>4657705</td><td></td></tr><tr><td>20 G 11/2`` Needles</td><td>4657519</td><td></td><td></td></tr><tr><td>Small scissor</td><td>WPI, Sarasota, USA</td><td>501263</td><td></td></tr><tr><td>Tweezers #5, 0.1 x 0.06 mm tip</td><td>WPI, Sarasota, USA</td><td>500342</td><td></td></tr><tr><td>Oxygen gas (medical grade O&lt;sub&gt;2&lt;/sub&gt;)</td><td>Linde, Munich, Germany</td><td></td><td></td></tr><tr><td>Carbogen gas (95 % O&lt;sub&gt;2&lt;/sub&gt;, 5 % CO&lt;sub&gt;2&lt;/sub&gt;)</td><td>Linde, Munich, Germany</td><td></td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich, St. Louise, USA</td><td>7647-14-5</td><td></td></tr><tr><td>KCl</td><td>Sigma-Aldrich, St. Louise, USA</td><td>746436</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich, St. Louise, USA</td><td>746495</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich, St. Louise, USA</td><td>NIST200B</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich, St. Louise, USA</td><td>51558</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma-Aldrich, St. Louise, USA</td><td>S5761</td><td></td></tr><tr><td>D(+)-Glucose</td><td>Sigma-Aldrich, St. Louise, USA</td><td>G8270</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich, St. Louise, USA</td><td>M7506</td><td></td></tr><tr><td>NaOH</td><td>Sigma-Aldrich, St. Louise, USA</td><td>S8045</td><td></td></tr><tr><td>Cyanoacrylate glue</td><td>Henkel, D&amp;uuml;sseldorf, Germany</td><td></td><td></td></tr><tr><td>Low-melt Agarose</td><td>Roth, Karlsruhe, Germany</td><td>6351.2</td><td></td></tr><tr><td>Heparin-sodium-25000 I.E./5 mL</td><td>Ratiopharm, Ulm, Germany</td><td></td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM), high glucose, GlutaMAX</td><td>ThermoScientific, Waltham, USA</td><td>10566016</td><td></td></tr><tr><td>SEC-10LX Amplifier</td><td>npi electronic GmbH, Tamm, Germany</td><td>SEC-10LX</td><td></td></tr><tr><td>EPC 9</td><td>HEKA Elektronik GmbH, Lambrecht, Germany</td><td></td><td></td></tr><tr><td>Zeiss Axiovert 200</td><td>Zeiss, Oberkochen, Germany</td><td></td><td></td></tr><tr><td>Low magnification Micromanipulator</td><td>Narashige, Tokyo, Japan</td><td>Nm-3</td><td></td></tr><tr><td>High magnification, three-axis micromanipulator</td><td>Narashige, Tokyo, Japan</td><td>MHW-3</td><td></td></tr><tr><td>Peristaltic perfusion pump</td><td>Multi Channel Systems, Reutlingen, Germany</td><td>PPS2</td><td></td></tr><tr><td>2-channel temperature controller</td><td>Multi Channel Systems, Reutlingen, Germany</td><td>TCO02</td><td></td></tr><tr><td>Square pulse stimulator</td><td>Natus Europe GmbH, Planegg, Germany</td><td>Grass SD9</td><td></td></tr><tr><td>Glass capillaries</td><td>WPI, Sarasota, USA</td><td>1B150F-1</td><td></td></tr></tbody></table>

murine short axis ventricular heart slices for electrophysiological studies

Fare kardiyomiyositleri , in vitro kardiyak fizyoloji çalışmaları ve yeni tedavi stratejileri için yoğun şekilde kullanılmaktadır. Bununla birlikte, ayrışmış kardiyomiyositlerin çok hücreli preparatları, kalbin hem mekanik hem de elektrofizyolojik özelliklerini etkileyen kardiyomiyositlerin, miyositlerin ve hücre dışı matrisin in vivo yapısının kompleksini temsil etmez. Burada, korunmuş in vivo doku yapısı ile yetişkin fare kalplerindeki canlı ventriküler dilimleri hazırlamak için bir teknik ve elektrofizyolojik kayıtlara uygunluğunu göstermek için bir teknik açıklanmaktadır. Kalbin eksizyonundan sonra, ventriküller atriyumdan ayrılır, 2,3-butandion monoksim içeren Ca2 + içermeyen çözelti ile perfüzyonlanır ve% 4&#39;lük düşük eriyikli agaroz blok içine gömülür. Blok titreşen bir bıçakla mikrotom üzerine yerleştirilir ve 150-400 μm kalınlığa sahip doku dilimleri titreşimi serbest bırakarak hazırlanır60 Hz&#39;de bıçak sıklığı ve bıçağı mümkün olduğunca yavaş ileri hareket ettirerek. Dilimlerin kalınlığı diğer uygulamalara bağlıdır. Dilimler buz soğukluğundaki Tyrode çözeltisinde 0.9 mM Ca 2+ ve 2,3-butandion monoksim (BDM) ile 30 dakika süreyle saklanır. Ardından, BDM&#39;yi yıkamak için dilimler 37 ° C DMEM&#39;ye 30 dakika boyunca aktarılır. Dilimler, kesici elektrodlar veya mikro elektrot dizileri ile elektrofizyolojik çalışmalar için, kontraktil fonksiyonu analiz etmek için güç ölçümleri için veya nakledilen kök hücre kaynaklı kardiyomiyositlerin ve konakçı doku arasındaki etkileşimi araştırmak için kullanılabilir. Keskin elektrot kayıtları için, bir dilim, ters çevrilmiş mikroskopun ısıtma plakasındaki 3 cm&#39;lik hücre kültürü çanağına yerleştirilir. Dilim bir tek kutuplu elektrot ile uyarılır ve dilim içindeki kardiyomiyositlerin hücre içi eylem potansiyelleri keskin bir cam elektrot ile kaydedilir.

Miyokard enfarktüsü kardiyomiyositlerin neredeyse geri döndürülemez bir kaybına yol açar. Eksojen kardiyak rejenerasyon için kök hücre kaynaklı kardiyomiyositleri kullanarak hücre replasman tedavisi umut verici bir tedavi yaklaşımıdır. Transplante hücrelerin elektriksel entegrasyonu ve olgunlaşması, hücre replasman tedavisinin güvenilirliği ve etkinliği için çok önemlidir. Entegrasyon ve olgunlaşmay?...

Watch this Scientific Journal Video about Murine Short Axis Ventricular Heart Slices for Electrophysiological Studies at JoVE.com

Murine Short Axis Ventricular Heart Slices for Electrophysiological Studies

Burada, yetişkin farelerden canlı ventriküler dilimlerin hazırlanmasını ve keskin elektrodun hareket potansiyel kayıtları için kullanımlarını açıklıyoruz. Bu çok hücreli müstahzarlar , in vitro olarak elektrofizyolojik ve farmakolojik çalışmalar için değerli bir model oluşturan, korunmuş bir in vivo doku yapısı sağlar.

Elektrofizyolojik Çalışmalar İçin Murin Kısa Eksenli Ventriküler Kalp Dilimleri

Murine cardiomyocytes have been extensively used for in vitro studies of cardiac physiology and new therapeutic strategies. However, multicellular ...

murine-short-axis-ventricular-heart-slices-for-electrophysiological

Research

JoVE Journal

Medicine

7.3K Görüntüleme.  University of Cologne.  Burada, yetişkin farelerden canlı ventriküler dilimlerin hazırlanmasını ve keskin elektrodun hareket potansiyel kayıtları için kullanımlarını açıklıyoruz. Bu çok hücreli müstahzarlar , in vitro olarak elektrofizyolojik ve farmakolojik çalışmalar için değerli bir model oluşturan, korunmuş bir in vivo doku yapısı sağlar. 

Video: Murine Short Axis Ventricular Heart Slices for Electrophysiological Studies

Preparation of Parasagittal Slices for the Investigation of Dorsal-ventral Organization of the Rodent Medial Entorhinal Cortex

Computation in the brain relies on neurons responding appropriately to their synaptic inputs. Neurons differ in their complement and distribution of membrane ion channels that determine how they respond to synaptic inputs. However, the relationship between these cellular properties and neuronal function in behaving animals is not well understood. One approach to this problem is to investigate topographically organized neural circuits in which the position of individual neurons maps onto information they encode or computations they carry out1. Experiments using this approach suggest principles for tuning of synaptic responses underlying information encoding in sensory and cognitive circuits2,3.
The topographical organization of spatial representations along the dorsal-ventral axis of the medial entorhinal cortex (MEC) provides an opportunity to establish relationships between cellular mechanisms and computations important for spatial cognition. Neurons in layer II of the rodent MEC encode location using grid-like firing fields4-6. For neurons found at dorsal positions in the MEC the distance between the individual firing fields that form a grid is on the order of 30 cm, whereas for neurons at progressively more ventral positions this distance increases to greater than 1 m. Several studies have revealed cellular properties of neurons in layer II of the MEC that, like the spacing between grid firing fields, also differ according to their dorsal-ventral position, suggesting that these cellular properties are important for spatial computation2,7-10.
Here we describe procedures for preparation and electrophysiological recording from brain slices that maintain the dorsal-ventral extent of the MEC enabling investigation of the topographical organization of biophysical and anatomical properties of MEC neurons. The dorsal-ventral position of identified neurons relative to anatomical landmarks is difficult to establish accurately with protocols that use horizontal slices of MEC7,8,11,12, as it is difficult to establish reference points for the exact dorsal-ventral location of the slice. The procedures we describe enable accurate and consistent measurement of location of recorded cells along the dorsal-ventral axis of the MEC as well as visualization of molecular gradients2,10. The procedures have been developed for use with adult mice (&gt; 28 days) and have been successfully employed with mice up to 1.5 years old. With adjustments they could be used with younger mice or other rodent species. A standardized system of preparation and measurement will aid systematic investigation of the cellular and microcircuit properties of this area.

We describe procedures for preparation and electrophysiological recording from brain slices that maintain the dorsal-ventral axis of the medial entorhinal cortex (MEC). Because neural encoding of location follows a dorsal-ventral organization within the MEC, these procedures facilitate investigation of cellular mechanisms important for navigation and memory.

Kemirgen Medial entorinal Korteksin dorsal-ventral Örgütü Soruşturma parasagital Dilimleri hazırlanması

Computation in the brain relies on neurons responding appropriately to their synaptic inputs. Neurons differ in their complement and distribution of ...

Nörobilim

The Preparation of Oblique Spinal Cord Slices for Ventral Root Stimulation

Electrophysiological recordings from spinal cord slices have proven to be a valuable technique to investigate a wide range of questions, from cellular to network properties. We show how to prepare viable oblique slices of the spinal cord of young mice (P2 - P11). In this preparation, the motoneurons retain&#160;their axons coming out from the ventral roots of the&#160;spinal cord. Stimulation of these axons elicits back-propagating action potentials invading the motoneuron&#160;somas and exciting the motoneuron&#160;collaterals within the spinal cord. Recording of antidromic action potentials is an immediate, definitive and elegant way to characterize motoneuron identity, which surpasses&#160;other identification methods. Furthermore, stimulating the motoneuron collaterals is a simple and reliable way to excite the collateral targets of the motoneurons within the spinal cord, such as other motoneurons or Renshaw cells. In this protocol, we present antidromic recordings from the motoneuron somas as well as Renshaw cell excitation, resulting from ventral root stimulation.

We show how to prepare oblique slices of the spinal cord in young mice. This preparation allows for the stimulation of the ventral roots.

Ventral Kök Stimulation için eğik Omurilik Dilimleri hazırlanması

Electrophysiological recordings from spinal cord slices have proven to be a valuable technique to investigate a wide range of questions, from cellular to ...

Preparation of Horizontal Slices of Adult Mouse Retina for Electrophysiological Studies

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in these preparations is perpendicular to the retinal surface, making it ideal for the study of radially oriented neurons like photoreceptors and bipolar cells. However, the large dendritic arbors of horizontal cells, wide-field amacrine cells, and ganglion cells are mostly truncated, leaving markedly reduced synaptic activity in these cells. Whereas ganglion cells and displaced amacrine cells can be studied in a whole-mounted preparation of the retina, horizontal cells and amacrine cells located in the inner nuclear layer are only poorly accessible for electrodes in whole retina tissue.
To achieve maximum accessibility and synaptic integrity, we developed a horizontal slice preparation of the mouse retina, and studied signal transmission at the synapse between photoreceptors and horizontal cells. Horizontal sectioning allows&#160;(1) easy and unambiguous visual identification of horizontal cell bodies for electrode targeting, and (2) preservation of the extended horizontal cell dendritic fields, as a prerequisite for intact and functional cone synaptic input to horizontal cell dendrites.
Horizontal cells from horizontal slices exhibited tonic synaptic activity in the dark, and they responded to brief flashes of light with a reduction of inward current and diminished synaptic activity. Immunocytochemical evidence indicates that almost all cones within the dendritic field of a horizontal cell establish synapses with its peripheral dendrites. The horizontal slice preparation is therefore well suited to study the physiological properties of horizontally extended retinal neurons as well as sensory signal transmission and integration across selected synapses.

We developed a horizontal slice preparation of the adult mammalian retina with the plane of sectioning parallel to the retinal surface. Dendritic fields of nonradially oriented retinal neurons were not truncated, allowing the study of signal processing with patch-clamp and imaging techniques.

Elektrofizyolojik Çalışmalar Yetişkin Fare Retina Yatay Dilimleri hazırlanması

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in ...

Preclinical Cardiac Electrophysiology Assessment by Dual Voltage and Calcium Optical Mapping of Human Organotypic Cardiac Slices

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between animal and clinical trials. Numerous animal and cell models have been used to examine the effects of drugs, yet these responses often differ in humans. Human cardiac slices offer an advantage for drug testing in that they are directly derived from viable human hearts. In addition to having preserved multicellular structures, cell-cell coupling, and extracellular matrix environments, human cardiac tissue slices can be used to directly test the effect of innumerable drugs on adult human cardiac physiology. What distinguishes this model from other heart preparations, such as whole hearts or wedges, is that slices can be subjected to longer-term culture. As such, cardiac slices allow for studying the acute as well as chronic effects of drugs. Furthermore, the ability to collect several hundred to a thousand slices from a single heart makes this a high-throughput model to test several drugs at varying concentrations and combinations with other drugs at the same time. Slices can be prepared from any given region of the heart. In this protocol, we describe the preparation of left ventricular slices by isolating tissue cubes from the left ventricular free wall and sectioning them into slices using a high precision vibrating microtome. These slices can then either be subjected to acute experiments to measure baseline cardiac electrophysiological function or cultured for chronic drug studies. This protocol also describes dual optical mapping of cardiac slices for simultaneous recordings of transmembrane potentials and intracellular calcium dynamics to determine the effects of the drugs being investigated.

This protocol describes the procedure for sectioning and culturing human cardiac slices for preclinical drug testing and details the use of optical mapping for recording transmembrane voltage and intracellular calcium signals simultaneously from these slices.

İnsan Organotipik Kardiyak Dilimlerinin Çift Voltaj ve Kalsiyum Optik Haritalaması ile Preklinik Kardiyak Elektrofizyoloji Değerlendirmesi

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between ...

Biyomühendislik

Slicing and Culturing Pig Hearts under Physiological Conditions

Many novel drugs fail in clinical studies due to cardiotoxic side effects as the currently available in vitro assays and in vivo animal models poorly predict human cardiac liabilities, posing a multi-billion-dollar burden on the pharmaceutical industry. Hence, there is a worldwide unmet medical need for better approaches to identify drug cardiotoxicity before undertaking costly and time consuming &#39;first in man&#39; trials. Currently, only immature cardiac cells (human induced pluripotent stem cell-derived cardiomyocytes [hiPSC-CMs]) are used to test therapeutic efficiency and drug toxicity as they are the only human cardiac cells that can be cultured for prolonged periods required to test drug efficacy and toxicity. However, a single cell type cannot replicate the phenotype of the complex 3D heart tissue which is formed of multiple cell types. Importantly, the effect of drugs needs to be tested on adult cardiomyocytes, which have different characteristics and toxicity responses compared to immature hiPSC-CMs. Culturing human heart slices is a promising model of intact human myocardium. This technology provides access to a complete multicellular system that mimics the human heart tissue and reflects the physiological or pathological conditions of the human myocardium. Recently, through optimization of the culture media components and the culture conditions to include continuous electrical stimulation at 1.2 Hz and intermittent oxygenation of the culture medium, we developed a new culture system setup that preserves viability and functionality of human and pig heart slices for 6 days in culture. In the current protocol, we are detailing the method for slicing and culturing pig heart as an example. The same protocol is used to culture slices from human, dog, sheep, or cat hearts. This culture system has the potential to become a powerful predictive human in situ model for acute cardiotoxicity testing that closes the gap between preclinical and clinical testing results.

This protocol describes how to slice and culture heart tissue under physiological conditions for 6 days. This culture system could be used as a platform for testing the efficacy of novel heart failure therapeutics as well as reliable testing of acute cardiotoxicity in a 3D heart model.

Fizyolojik Koşullar altında Domuz Kalpleri Dilimleme ve Culturing

Many novel drugs fail in clinical studies due to cardiotoxic side effects as the currently available in vitro assays and in vivo animal models poorly ...

Tıp

Dissection Techniques and Histological Sampling of the Heart in Large Animal Models for Cardiovascular Diseases

The standard gross examination and sampling are key elements in the reproducibility and success of experimental studies of cardiovascular diseases carried out in large animals. Considering the complex anatomy of the heart, interspecies differences, and the types of compensatory and pathological reactions, consistent protocols are challenging to implement. The utilization of multiple dissection protocols is usually adapted to suit the prosector's experience, and personal preference continues to be a source of experimental and interobserver variability. Here, the aim is to present the main anatomical features and landmarks, dissection protocols, and histological sampling standards of the heart in some commonly used species (including dogs, pigs, ruminants, and cats) as models of cardiovascular diseases.
Two standard gross examination protocols are presented here. First, the inflow-outflow method, which follows the physiological blood flow direction through the heart and large vessels (frequently used in dogs, ruminants, and pigs), and second, the four-chamber dissection technique (exemplified in cats). Both techniques can be adapted to any species in certain experimental circumstances. The sampling protocols include all the areas of interest (sinoatrial node, ventricles, interventricular septum, atria, valves, and aorta), and if properly carried out, improve both the reproducibility and reliability of experimental studies.

Due to the complex anatomy, a consistently reproducible dissection and harvesting protocol for cardiac samples can be challenging to implement. This manuscript presents the key elements of some standard cardiac dissection protocols, highlighting both the gross examination approaches and the sampling sites commonly used for histopathologic examination.

Diseksiyon Teknikleri ve Kardiyovasküler Hastalıklar için Büyük Hayvan Modellerinde Kalbin Histolojik Örneklemesi

The standard gross examination and sampling are key elements in the reproducibility and success of experimental studies of cardiovascular diseases carried ...

Preparation of Human Myocardial Tissue for Long-Term Cultivation

Cardiomyocyte cultivation has seen a vast number of developments, ranging from two-dimensional (2D) cell cultivation to iPSC derived organoids. In 2019, an ex vivo way to cultivate myocardial slices obtained from human heart samples was demonstrated, while approaching in vivo condition of myocardial contraction. These samples originate mostly from heart transplantations or left-ventricular assist device placements. Using a vibratome and a specially developed cultivation system, 300 &#181;m thick slices are placed between a fixed and a spring wire, allowing for stable and reproducible cultivation for several weeks. During cultivation, the slices are continuously stimulated according to individual settings. Contractions can be displayed and recorded in real-time, and pharmacological agents can be readily applied. User-defined stimulation protocols can be scheduled and performed to assess vital contraction parameters like post-pause-potentiation, stimulation threshold, force-frequency relation, and refractory period. Furthermore, the system enables a variable pre- and afterload setting for a more physiological cultivation.
Here, we present a step-by-step guide on how to generate a successful long-term cultivation of human left ventricular myocardial slices, using a commercial biomimetic cultivation solution.

We present a protocol for ex vivo cultivation of human ventricular myocardial tissue. It allows for detailed analysis of contraction force and kinetics, as well as the application of pre- and afterload to mimic the in vivo physiological environment more closely.

Uzun Süreli Yetiştirme için İnsan Miyokard Dokusunun Hazırlanması

Cardiomyocyte cultivation has seen a vast number of developments, ranging from two-dimensional (2D) cell cultivation to iPSC derived organoids. In 2019, ...

Isolation of Human Ventricular Cardiomyocytes from Vibratome-Cut Myocardial Slices

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly isolated by coronary perfusion with digestive enzymes. However, isolating human cardiomyocytes is challenging because human myocardial specimens usually do not allow for coronary perfusion, and alternative isolation protocols result in poor yields of viable cells. In addition, human myocardial specimens are rare and only regularly available at institutions with on-site cardiac surgery. This hampers the translation of findings from animal to human cardiomyocytes. Described here is a reliable protocol that enables efficient isolation of ventricular myocytes from human and animal myocardium. To increase the surface-to-volume ratio while minimizing cell damage, myocardial tissue slices 300 &#181;m thick are generated from myocardial specimens with a vibratome. Tissue slices are then digested with protease and collagenase. Rat myocardium was used to establish the protocol and quantify yields of viable, calcium-tolerant myocytes by flow-cytometric cell counting. Comparison with the commonly used tissue-chunk method showed significantly higher yields of rod-shaped cardiomyocytes (41.5 &#177; 11.9 vs. 7.89 &#177; 3.6%, p &#60; 0.05). The protocol was translated to failing and non-failing human myocardium, where yields were similar as in rat myocardium and, again, markedly higher than with the tissue-chunk method (45.0 &#177; 15.0 vs. 6.87 &#177; 5.23 cells/mg, p &#60; 0.05). Notably, with the protocol presented it is possible to isolate reasonable numbers of viable human cardiomyocytes (9&#8211;200 cells/mg) from minimal amounts of tissue (&#60;50 mg). Thus, the method is applicable to healthy and failing myocardium from both human and animal hearts. Furthermore, it is possible to isolate excitable and contractile myocytes from human tissue specimens stored for up to 36 h in cold cardioplegic solution, rendering the method particularly useful for laboratories at institutions without on-site cardiac surgery.

Presented is a protocol for the isolation of human and animal ventricular cardiomyocytes from vibratome-cut myocardial slices. High yields of calcium-tolerant cells (up to 200 cells/mg) can be obtained from small amounts of tissue (&#60;50 mg). The protocol is applicable to myocardium exposed to cold ischemia for up to 36 h.

Vibratome-Cut Miyokardiyal Dilimlerden İnsan Ventriküler Kardiyomiyositlerin İzolasyon

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly ...

Biyoloji

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

Ca2+ geçici ve L-tipi kalsiyum akımının eşzamanlı ölçümleri için yüksek kaliteli murin atriyal ve ventriküler miyositlerin izolasyonu

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...

Generation of Porcine Ventricular Slices: A Vibratome-based Technique to Prepare Ultra-thin Slices of Pig Heart Tissue

Source: Ou, Q. et. al., <a href="https://www.jove.com/v/60913">Slicing and Culturing Pig Hearts under Physiological Conditions.</a> J. Vis. Exp. (2020)
This video demonstrates a vibratome-based procedure to generate viable, ultra-thin sections of pig heart. The prepared tissue slices can be cultured and used as model systems for pharmacological testing.

Domuz Ventriküler Dilimlerinin Oluşturulması: Ultra İnce Domuz Kalp Dokusu Dilimleri Hazırlamak İçin Vibratom Bazlı Bir Teknik

Source: Ou, Q. et. al., Slicing and Culturing Pig Hearts under Physiological Conditions. J. Vis. Exp. (2020)
This video demonstrates a vibratome-based ...