This work was supported by National Medical Research Council (NMRC)/Cooperative Basic Research Grant (CBRG)/0078/2014.

Bu çalışma Singapur Kurumsal Hayvan Bakımı ve Kullanımı Komitesi (onaylanan protokole hayır. R15-0225) Ulusal Üniversitesi ile uyumludur. Hayvan Modeli 1. Cerrahi Hazırlık <ol><li> Gemi kanülasyonlar <ol><li> Erkek bir Sprague-Dawley sıçanları anestezi - ağırlığında (6 7 haftalık), (203 ± 20) intraperitonal yoluyla ketamin (37.5 mg / ml) ve ksilazin (5 mg / ml) kokteyli ile gr (ip) enjeksiyonu (2 ml / kg) . İğneyi tekrarlamak ya da enjeksiyondan sonra şırınga çıkarmayın. </li><li> hayvana anestezi yapıldıktan sonra (kıstırma ayak ile teyit), 37 ° C de vücut ısısını korumak için bir ısıtma pedi üzerine yerleştirin. Yavaşça skapula, ön serviks, alt karın, medial arka bacak ve skrotal kese saç tıraş. Yavaşça yapışkanlı kağıt bantlar kullanarak bacakları dizginlemek. </li><li> Mikrodiseksiyon makas ve açılı forseps ile izlerken kullanarak tüm cerrahi prosedürleri gerçekleştirinBir stereomikroskop. Ameliyat sırasında yaralanmayı önlemek için delinmeye karşı dayanıklı bir tepsiye tüm keskin cerrahi aletler yerleştirin. </li><li> kesi yapmadan önce tüm cerrahi siteleri alternatif iyot ve% 70 alkol ile 3 kez fırçalayın. 30 IU / ml heparin tuzlu su çözeltisi ile tüm kateterler yıkayın. </li><li> Sağ juguler ven üzerinde cerrahi makas kullanarak skapula üzerinde 1,5 cm orta hat cilt kesi - 1 yapın. juguler ven ortaya çıkarmak için künt diseksiyon ile fasya ayırın ve bir polietilen tüp (PE-50) 5-0 ipek sütür kullanılarak heparin tuzlu su ile dolu olan cannulate. Yan anestezi infüze gerektiğinde (1/3 başlangıç ​​dozunun 1/2, damar içine (IV)) cerrahi ve deney süresince. </li><li> havayolu açıklığını korumak için trakeostomi gerçekleştirin. anterior servikal bölgede 1,5 cm kesi - 1 yapın. yerine kateteri sabitlemek için 2-0 ipek dikişlerle bir polietilen tüp (PE-205) kullanılarak trakea cannulate. </li><li> Kan basıncını izlemekFemoral arter kanülasyonu yoluyla. arka bacak sol medial yüzeyi 1.5 cm&#39;lik kesi - 1 yapın. künt diseksiyon ile femoral arter ayırın. heparin tuz çözeltisi 5-0 ipek sütür kullanılarak doldurulmuş bir polietilen boru (PE-10), femoral arter kanüle. </li></ol></li><li> Cremaster kas Hazırlama ve Akış Görselleştirme <ol><li> bunu genişletmek için skrotal kese apeks yoluyla 5-0 ipek sütür yerleştirin. skrotal kese ventral yüzeyi boyunca bir kesi yapmak. maruz kas, düzenli sıcak izotonik solüsyonu (pH 7.4 37 ° C) uygulanır. </li><li> dikkatle ve iyice bağ dokuları çevreleyen pamuk uçlu aplikatör kullanarak kaldırın. </li><li> cremaster kas apeks yoluyla 5-0 ipek sütür yerleştirin. aynı uzunluktaki iki parçaya dikiş kesin ve her iki tarafta bir düğüm. İki düğüm arasındaki kas kesin ve hafifçe dikiş çekerek özelleştirilmiş şeffaf pleksiglas platformu üzerine germek. düzeltmekmavi yapışma ile platform üzerine sütür sonu. NOT: bağ dokuları çevreleyen iyice çıkarılması optimum görüntü kontrastını elde önemlidir. </li><li> 5 ila 6 tespitler yapılana kadar adımı tekrarlayın 1.2.3. Dikkatle yüksek sıcaklık koter kullanılarak Epididimden cremaster kasını çıkarın. doku kaybını önlemek için maruz kas sıcak izotonik Superfuse. <ol><li> gazlı katlanmış parçalar ile cremaster kasını Surround. polivinil film ile maruz kas örtün. Film ile gazlı bez parçaları su daldırma mikroskop objektif (Şekil 1A) için sıcak izotonik çözüm tutmak için sığ bir havza oluşturur. </li></ol></li><li> Intravital mikroskobu (Şekil 1C) hayvan sahneye hayvan aktarın. Sürekli basıncı izleme (Şekil 1E) için fizyolojik bir veri toplama sistemine arteriyel kanülasyon bağlayın. </li><li> kas temperat korumakHayvan platforma (Şekil 1B) altındaki bağlı bir ısıtma elemanı ile 35 ° C &#39;de ure. Isıtma elemanı (Şekil 1D) güç kontrolörü negatif geribildirim sağlamak için kas yanında bir sıcaklık probu yerleştirin. </li><li> 15 dk çevre ile dengelenmesi için sahnede hayvan bırakın. </li><li> 40X su daldırma objektif ve uzun çalışma kondansatör ile bir intravital mikroskop altında kan akışını gözünüzde canlandırın. </li><li> dallanmamış arteriolden RBC çekirdek arasında net bir görüntü odak ve kontrast dayalı (&lt;60 mikron) seçin, CFL ve damar duvarları, sırayla kan damarı çap düzleminde mikroskop odaklanmak. dikey damar duvarını hizalamak için mikroskop üzerine monte kamera döndürün. </li><li> 1 saniye 3000 / sn kare hızında yüksek hızlı video kamera kullanılarak kan akışını kaydedin. görüntü kalitesini korumak için sıkıştırılmamış 8-bit gri tonlama AVI biçimi olarak kaydedilen video kaydedin. NOT:<html> / Sn 3.000 kare en az kayıt hızı flüoresan ölçümü fizyolojik arteriolar akış koşulları altında en az bir defa her RBC ile gerçekleştirilebilir sağlamak için önerilir. </li><li> Eritrosit ve plazma arasındaki kontrastı arttırmak için 510 nm - 310 394 nm ve spektral bandpass dalga boyunda tepe şanzıman ile mavi filtre kullanın. NOT: mikroskobik ışık kaynağı (100 W halojen lamba) mavi filtreden geçen ışık spektrumu herhangi bir potansiyel doku hasarı önlemek için düşük ışık yoğunluğu olduğundan emin olun. </li><li> Deneyin sonunda, pentobarbital sodyum, aşırı dozda hayvan euthanize. </li></ol></li></ol> 2. Görüntü Analizi <ol><li> Flüoresan genişliği ölçümü için Ön işleme <ol><li> MATLAB açın ve &#39;CFL_pre.m&#39; dosyasını çalıştırın. (Bu ve diğer MATLAB dosyaları bulunabilirip &quot;&gt; Yan MATLAB Arşivi.) </li><li> analiz etmek, video dosyasını seçmek için &#39;Açık dosya&#39; tıklayın. </li><li> dikey damar duvarları hizalamak için &#39;rotasyon&#39; sürgüsünü ayarlayın. NOT: Kullanıcılar &#39;Izgara On&#39; radyo düğmesini seçerek damar uyum için yardımcı ızgara çizgileri görüntüler ve &#39;Zoom&#39; kaydırıcıyı kaydırarak görüntüyü yakınlaştırma düzeyini ayarlayabilirsiniz. </li><li> damar uyumu onaylamak için &#39;Onayla Düzenlenmesi&#39; butonuna tıklayın. </li><li> Faiz (ROI) bölgeyi tanımlamak için &#39;Set ROI ekine&#39; butonuna tıklayın. hizalanmış görüntü bir pop-up penceresi görüntülenir. görüntü üzerinde dikdörtgen hedefi ayarlayın ve çift ROI onaylamak için tıklayın. Görüntünün kırpma gerekli değilse bu adımı atlayın. NOT: Sadece gemiden CFL genişliği analiz etmek ROI tek bir gemi bulunmaktadır. Gerekirse, orijinal forma görüntüyü geri yüklemek için &#39;Reset Resim&#39; düğmesine tıklayın. </li><li> Tıkla9; &#39;(bmp&#39; biçiminde ardışık bit haritası görüntüleri 8-bit gri tonlama) içine tüm düzenlenmiş video kareleri ayıklamak için düğmeye &#39;Images ayıklayın. çıkarılan görüntüler seçilen video dosyası ile aynı adı taşıyan klasörde bulunabilir. </li></ol></li><li> Flüoresan genişliğinin ölçümü <ol><li> MATLAB açın ve &#39;CFL_measure.m&#39; dosyasını çalıştırın. </li><li> çıkarılan görüntüleri içeren klasörü seçmek için &#39;Klasör seç&#39; seçeneğini tıklayın. </li><li> görüntüleri içeren klasöre tıklayın ve &#39;Klasör seç&#39; seçeneğini tıklayın. klasördeki ilk görüntü karesi yüklenen ve &#39;Resim Histogram&#39; panelinde gri yoğunluk Histogram ile birlikte, &#39;Gri tonlama görüntü&#39; panelinde gösterilir. </li><li> analizi, aksi takdirde ilk görüntü karesi seçilecektir gerçekleştirmek için liste kutusundan istenen görüntü çerçevesini seçin. </li><li> yerde belirlenir görüntüde, iç damar duvarını tanımlamak için &#39;Gemi Duvarlar Bul&#39; tıklayın neredekaranlıktan ışık yoğunluğu profil tepe transit iki piksel üzerinde ışık. </li><li> &#39;Tuz ve biber&#39; gürültüyü azaltmak için görüntüye medyan filtre uygulamak için &#39;Medyan Filtre&#39; kontrol edin. </li><li> görüntü kontrastını arttırmak için dijital görüntü yoğunlukları ayarlamak için &#39;Otomatik Kontrast&#39; kontrol edin. </li><li> τ üzerinde gri düzeyleri (CFL) ve τ altında gri düzeyleri (RBC çekirdek) siyah pikseller beyaz piksel - iki sınıfa gri düzeyleri böler otomatik olarak bir eşik değeri (τ) belirleyen liste kutusunda bir eşik algoritmasını seçin. NOT: Otomatik-eşikleme algoritması hiçbiri uygun bir görüntü eşikleme sağlıyorsa alternatif bir yöntem olarak, manuel eşikleme kullanın. &#39;Elle&#39; radyo düğmesini tıklayın ve manuel eşikleme değeri tanımlamak için kaydırma çubuğunu ayarlayın. </li><li> (Çözünürlük, CFL genişlikleri mekansal değişimini ölçen &#39;piksel çözünürlük&#39; kutusuna piksel çözünürlüğünü girmek içinBu deney düzeneği ile 0.42 mm / piksel) idi. </li><li> CFL genişlikleri mekansal değişimi elde etmek için &#39;Hesapla&#39; düğmesine tıklayın. bir tablo formatında CFL genişliği veri vermek için &#39;İhracat .csv tıklayın. </li><li> kare hızı bilgilerini, geminin boyunca belirli bir analiz hattında CFL genişlikleri zamansal değişimini ölçen &#39;Temporal Varyasyonu&#39; radyo düğmesini tıklayın ve girmek için (bu deney düzeneği kullanılan kare hızını 3000 kare / sn idi). </li><li> sırasıyla, &#39;Frame başlat&#39; ve &#39;Son Frame&#39; kutulara ilk kareyi ve analiz için görüntülerin son karesini girin. </li><li> &#39;Analiz Line&#39; kaydırma çubuğunu kaydırarak geminin boyunca analiz hattının pozisyonunu seçin. &#39;Grayscale görüntü&#39; ve &#39;İkili görüntü&#39; hem gösterilmiştir analiz hattının, konumunu onaylayın. </li><li> CFL genişlikleri zamansal değişimini elde etmek için &#39;hesapla&#39; tıklayın. click &#39;İhracat .csv&#39; bir tablo formatında CFL genişliği veri vermek için. </li></ol></li></ol>

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The authors declare no competing financial interests.

CFL genişlik ölçüm mikrosirkülasyonun hemodinamik daha iyi anlaşılması için önemlidir. Özel olarak, flüoresan genişlikleri ölçümü mezenterik 6&#39;da gerçekleştirilmiştir, 24 ve serebral 25 mikrodolaşımını spinotrapezius. In vivo flüoresan genişlikleri geleneksel ölçüm kaydedilen video çerçevelerinin elle muayene ile tahminlerine sınırlandırılmıştır. Manuel ölçümler görsel RBC çekirdek ve damar duvarlarının 15,16 sınırlar...

In vivo animal studies are instrumental to basic science for understanding human physiology and pathology. In particular, in vivo microhemodynamic studies can elucidate the potential impairment of microcirculatory functions altered by abnormal rheological conditions of blood. A number of previous microhemodynamic studies1 have used the rat cremaster muscle model for visualizing microvascular blood flow. The cremaster muscle is a thin layer of striated muscle surrounding the testes. Thus, the blood flow in the muscle can be visualized with a trans-illumination microscope by means of surgical exposure. This enables us to acquire the in vivo blood flow images without the use of any fluorescence or contrast agents. In addition, the entire blood perfusion of the muscle network can be controlled by reducing the upstream blood flow with abdominal aorta occlusion2. Owing to these advantages, the cremaster muscle model has been widely used to investigate the formation of cell-free layer (CFL) in microvessels1,3. 
The CFL width is a prominent hemodynamic parameter in microcirculation, which has been of great interest for its important roles in regulating microcirculatory functions. The CFL is formed by the shear-induced transverse inward migration of red blood cells (RBCs) towards the flow center4. Consequently, this migration leads to the depletion of RBCs near the vessel walls, eventually resulting in a cell-free plasma layer. Accordingly, the parietal CFL naturally becomes a diffusion barrier to oxygen (O2) delivery from the RBC core to the tissues, and to the scavenging of nitric oxide (NO) by the RBCs5,6. In addition, the production of NO can also be modulated by the dynamic variations of the CFL width7,8. Therefore, the roles of the CFL in both gas transport and the regulation of homeostasis in microcirculation need to be fully ascertained to better understand blood flow in microcirculation. Recent studies have focused on bridging the hemodynamics and gas transport functions of the CFL in the microcirculation9-12. Furthermore, a separate set of studies has also investigated how the pathological elevation in RBC aggregation modulates CFL formation and its effect on O2 and NO bioavailability in tissues13,14. 
The roles of the CFL become more significant in microcirculation where the relative size of the CFL width to the vessel diameter is prominent. This necessitates an effective approach of quantifying the CFL in in vivo blood flow. Particularly, image acquisition and image analysis are the two key components determining the accuracy of CFL width measurement. Successful visualization of tissue blood flow should be preceded by an appropriate surgical preparation of the animal model. Additionally, a proper image analysis technique is needed to overcome the limitations of conventional manual measurements that are mostly induced by human errors15,16. With advancements in optical instrumentation and computing power for digital image processing, it is now possible to achieve a more accurate and consistent measurement of the CFL width17-19. Nonetheless, the accuracy of these measurements, being image-based, still ultimately depends on the quality of the images.
Therefore, this study explores the factors influencing the measurement of the in vivo CFL width. We focused particularly on demonstrating the surgical preparation and digital image analysis for measurements of the CFL width in arterioles of the rat cremaster muscle.

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			<td>.038&#34; OD X .023&#34; ID</td>
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			<td height="21" style="height:21px;width:251px;">PE-205 polyethene tube</td>
			<td style="width:167px;">Becton Dickinson</td>
			<td style="width:119px;">427446</td>
			<td>.082&#34; OD X .062&#34; ID</td>
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			<td height="21" style="height:21px;width:251px;">2-0 non-absorbable silk suture</td>
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			<td height="21" style="height:21px;width:251px;">5-0 non-absorbable silk suture</td>
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			<td height="21" style="height:21px;width:251px;">Sterile Cotton Gauze&#160;</td>
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			<td style="width:119px;">22-415-468</td>
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			<td style="width:167px;">Fisher Scientific</td>
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			<td height="21" style="height:21px;width:251px;">Dumont Forceps</td>
			<td style="width:167px;">Kent Scientific</td>
			<td style="width:119px;">INS14188</td>
			<td>Surgical instrument</td>
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			<td height="21" style="height:21px;width:251px;">Micro Dissecting forceps</td>
			<td style="width:167px;">Kent Scientific</td>
			<td style="width:119px;">INS15915</td>
			<td>Surgical instrument</td>
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			<td height="21" style="height:21px;width:251px;">Iris forceps 1x2 teeth</td>
			<td style="width:167px;">Kent Scientific</td>
			<td style="width:119px;">INS15917</td>
			<td>Surgical instrument</td>
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			<td height="21" style="height:21px;width:251px;">Vessel cannulation forceps</td>
			<td style="width:167px;">Kent Scientific</td>
			<td style="width:119px;">INS500377</td>
			<td>Surgical instrument</td>
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			<td height="21" style="height:21px;width:251px;">Micro scissor</td>
			<td style="width:167px;">Kent Scientific</td>
			<td style="width:119px;">INS14177</td>
			<td>Surgical instrument</td>
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			<td height="21" style="height:21px;width:251px;">Iris scissor</td>
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			<td style="width:167px;">Kent Scientific</td>
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			<td height="21" style="height:21px;width:251px;">Gemini cautery system</td>
			<td style="width:167px;">Braintree Scientific</td>
			<td style="width:119px;">GEM 5917</td>
			<td>Surgical instrument</td>
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visualization and quantification of the cell-free layer in arterioles of the rat cremaster muscle

The cell-free layer is defined as the parietal plasma layer in the microvessel flow, which is devoid of red blood cells. The measurement of the in vivo cell-free layer width and its spatiotemporal variations can provide a comprehensive understanding of hemodynamics in microcirculation. In this study, we used an intravital microscopic system coupled with a high-speed video camera to quantify the cell-free layer widths in arterioles in vivo. The cremaster muscle of Sprague-Dawley rats was surgically exteriorized to visualize the blood flow. A custom-built imaging script was also developed to automate the image processing and analysis of the cell-free layer width. This approach enables the quantification of spatiotemporal variations more consistently than previous manual measurements. The accuracy of the measurement, however, partly depends on the use of a blue filter and the selection of an appropriate thresholding algorithm. Specifically, we evaluated the contrast and quality of images acquired with and without the use of a blue filter. In addition, we compared five different image histogram-based thresholding algorithms (Otsu, minimum, intermode, iterative selection, and fuzzy entropic thresholding) and illustrated the differences in their determination of the cell-free layer width.

In vivo CFL görselleştirme hayvanın cerrahi hazırlıkları büyük ölçüde bağlıdır. Aşırı kan kaybı ya da genişletilmiş cerrahi süresi şok ve kan akımı sapmaları hayvan tabi olabilir. bir ısıtma yastığı yanı sıra cerrahi ve deney sırasında özelleştirilmiş bir platformu kullanarak doku sıcaklığı bakım da sıçan fizyolojik koşulları sağlamak için önemlidir. mikroskop sistemi bir 100 W halojen lamba kullanarak, hiç bir farkedilebilir doku hasarı da deneyin sonunda göz...

Watch this Scientific Journal Video about Visualization and Quantification of the Cell-free Layer in Arterioles of the Rat Cremaster Muscle at JoVE.com

Visualization and Quantification of the Cell-free Layer in Arterioles of the Rat Cremaster Muscle

This study demonstrates the surgical preparation of the rat cremaster muscle for the visualization of the in vivo cell-free layer. Considerable factors affecting the accuracy of the cell-free layer width measurement are discussed in this study.

Sıçan Cremaster&#39;ın Muscle arteriyollerde Hücre-serbest Katman Görselleştirme ve

The cell-free layer is defined as the parietal plasma layer in the microvessel flow, which is devoid of red blood cells. The measurement of the in vivo ...

visualization-quantification-cell-free-layer-arterioles-rat-cremaster

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

6.3K Görüntüleme.  National University of Singapore. This study demonstrates the surgical preparation of the rat cremaster muscle for the visualization of the in vivo cell-free layer. Considerable factors affecting the accuracy of the cell-free layer width measurement are discussed in this study.

Video: Visualization and Quantification of the Cell-free Layer in Arterioles of the Rat Cremaster Muscle

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

İyonlaştırıcı Radyasyon Cevabı γH2AX Foci Kantitasyonu

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Biyoloji

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

İskelet Kası progenitörlerin saflaştırılması

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

The Mouse Cremaster Muscle Preparation for Intravital Imaging of the Microcirculation

Throughout the body, the maintenance of homeostasis requires the constant supply of oxygen and nutrients concomitant with removal of metabolic by-products. This balance is achieved by the movement of blood through the microcirculation, which encompasses the smallest branches of the vascular supply throughout all tissues and organs. Arterioles branch from arteries to form networks that control the distribution and magnitude of oxygenated blood flowing into the multitude of capillaries intimately associated with parenchymal cells. Capillaries provide a large surface area for diffusional exchange between tissue cells and the blood supply. Venules collect capillary effluent and converge as they return deoxygenated blood towards the heart. To observe these processes in real time requires an experimental approach for visualizing and manipulating the living microcirculation.
The cremaster muscle of rats was first used as a model for studying inflammation using histology and electron microscopy post mortem1,2. 
The first in vivo report of the exposed intact rat cremaster muscle investigated microvascular responses to vasoactive drugs using reflected light3. However 
curvature of the muscle and lack of focused illumination limited the usefulness of this preparation. The major breakthrough entailed opening the muscle, 
detaching it from the testicle and spreading it radially as a flat sheet for transillumination under a compound microscope4. While shown to be a valuable 
preparation to study the physiology of the microcirculation in rats5 and hamsters6, the cremaster muscle in mice7 has proven particularly useful in dissecting 
cellular pathways involved in regulating microvascular function8-11 and real-time imaging of intercellular signaling12. 
The cremaster muscle is derived from the internal oblique and transverse abdominus muscles as the testes descend through the inguinal canal13. 
It serves to support (Greek: cremaster = suspender) and maintain temperature of the testes. As described here, the cremaster muscle is prepared as a thin flat sheet 
for outstanding optical resolution. With the mouse maintained at a stable body temperature and plane of anesthesia, surgical preparation involves freeing the muscle 
from surrounding tissue and the testes, spreading it onto transparent pedestal of silastic rubber and securing the edges with insect pins while irrigating it 
continuously with physiological salt solution. The present protocol utilizes transgenic mice expressing GCaMP2 in arteriolar endothelial cells. GCaMP2 is a 
genetically encoded fluorescent calcium indicator molecule12. Widefield imaging and an intensified charge-coupled device camera enable in vivo study of 
calcium signaling in the arteriolar endothelium.

A tissue preparation is described for visualization and experimental manipulation of the living microcirculation. In anesthetized male mice, the thin, highly vascularized cremaster muscle is prepared for intravital microscopy to study microvascular networks including arterioles, capillaries and venules. This preparation is readily adapted for rats and hamsters.

Mikrosirkülasyon intravital Görüntüleme Fare Cremaster&#39;ın kas Hazırlık

Throughout the body, the maintenance of homeostasis requires the constant supply of oxygen and nutrients concomitant with removal of metabolic ...

Imaging of Estrogen Receptor-&alpha; in Rat Pial Arterioles using a Digital Immunofluorescent Microscope

Many of estrogen's effects on vascular reactivity are mediated through interaction with estrogen receptors 1, 2, 3. Although two sub-types exist (estrogen receptor -&alpha; and &beta;),estrogen receptor-&alpha; has been identified in both the smooth muscle and in endothelial cells of pial arterial segments using fluorescent staining combined with confocal laser scanning microscopy 4. Furthermore, ER-&alpha; is located in the nuclei and in the cytoplasm of rat basilar arteries 5. The receptors are abundant and fluoresce brightly, but clear visualization of discrete groups of receptors is difficult likely due to the numbers located in many cell layers of pial vessel segments. Additionally, many reports using immunohistochemical techniques paired with confocal microscopy poorly detail the requirements critical for reproduction of experiments 6. Our purpose for this article is to describe a simple technique to optimize the staining and visualization of ER-&alpha; using cross-sectional slices of pial arterioles obtain from female rat brains. We first perfuse rats with Evans blue dye to easily identify surface pial arteries which we isolate under a dissecting microscope. Use of a cryostat to slice 8 &mu;m cross sections of the arteries allows us to obtain thin vessel sections so that different vessel planes are more clearly visualized. Cutting across the vessel rather than use of a small vessel segment has the advantage of easier viewing of the endothelial and smooth muscle layers. In addition, use of a digital immunofluorescent microscope with extended depth software produces clear images of ten to twelve different vessel planes and is less costly than use of a confocal laser scanning microscope.

Imaging of Estrogen Receptor-&#945; in Rat Pial Arterioles using a Digital Immunofluorescent Microscope

The goal of this article is to demonstrate a method to optimize immunofluorescent detection of estrogen receptor-&#945; (ER&#945;) in rat pial arterial slices using a digital immunofluorescent microscope.

Rat pial arteriollerde Östrojen Reseptör-α Dijital Immunofluorescent Mikroskobu kullanılarak görüntülenmesi

Many of estrogen's effects on vascular reactivity are mediated through interaction with estrogen receptors 1, 2, 3. Although two sub-types exist (estrogen ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

Floresan kullanarak mRNA Moleküllerin Görselleştirme ve Analiz<em&gt; Yerinde</emIn&gt; Hibridizasyon<em&gt; Saccharomyces cerevisiae</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

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 ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

Kalsiyum değerlendirilmesi Bozulmamış İskelet kas liflerinin içinde Sparks

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with real time imaging, it becomes feasible to determine permeability changes in individual pulmonary microvessels. Herein we describe steps to isolate rat lungs and perfuse them with autologous blood. Then, we outline steps to infuse fluorophores or agents via a microcatheter into a small lung region. Using these procedures described, we determined permeability increases in rat lung microvessels in response to infusions of bacterial lipopolysaccharide. The data revealed that lipopolysaccharide increased fluid leak across both venular and capillary microvessel segments. Thus, this method makes it possible to compare permeability responses among vascular segments and thus, define any heterogeneity in the response. While commonly used methods to define lung permeability require postprocessing of lung tissue samples, the use of real time imaging obviates this requirement as evident from the present method. Thus, the isolated lung preparation combined with real time imaging offers several advantages over traditional methods to determine lung microvascular permeability, yet is a straightforward method to develop and implement.

The isolated blood-perfused lung preparation makes it feasible to visualize microvessel networks on the lung surface. Here we describe an approach to quantify permeability of single microvessels in isolated lungs using real time fluorescence imaging.

İzole Kan-perfüze Sıçan Akciğer Hazırlık Tek Mikrodamar Geçirgenliği miktarının

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with ...

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.

Murin Koroner Damar düz kas hücrelerinin izolasyonu

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

A Rapid Automated Protocol for Muscle Fiber Population Analysis in Rat Muscle Cross Sections Using Myosin Heavy Chain Immunohistochemistry

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle composition. Various time-consuming methods have traditionally been used to study fiber populations in many fields of research. However, recently developed immunohistochemical methods based on myosin heavy chain protein expression provide a quick alternative to identify multiple fiber types in a single section. Here, we present a rapid, reliable and reproducible protocol for improved staining quality, allowing automatic acquisition of whole cross sections and automatic quantification of fiber populations with ImageJ. For this purpose, embedded skeletal muscles are cut in cross sections, stained using myosin heavy chains antibodies with secondary fluorescent antibodies and DAPI for cell nuclei staining. Whole cross sections are then scanned automatically using a slide scanner to obtain high-resolution composite pictures of the entire specimen. Fiber population analyses are subsequently performed to quantify slow, intermediate and fast fibers using an automated macro for ImageJ. We have previously shown that this method can identify fiber populations reliably to a degree of &#177;4%. In addition, this method reduces inter-user variability and time per analyses significantly using the open source platform ImageJ.

Here, we present a protocol for rapid muscle fiber analyses, which allows improved staining quality, and thereby automatic acquisition and quantification of fiber populations using the freely available software ImageJ.

Miyosin Ağır Zincir İmünohistokimya kullanma Sıçan kas Çapraz Bölüm kas Elyaf Nüfus Analizi İçin Hızlı Otomatik Protokolü

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle ...

Sıçan Cremaster'ın Muscle arteriyollerde Hücre-serbest Katman Görselleştirme ve

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