ويدعم البحوث في المختبر المؤلفين من المعاهد الوطنية للصحة منح R37 - HL041026 ، R01 - R01 HL086483 وHL056786 - (SSS) وHL097463 - F32 و- T32 AR048523 (PB) من خدمة الولايات المتحدة الصحة العامة.

<ol>
	<li>Majno, G., Palade, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+inflammation.+1.+The+effect+of+histamine+and+serotonin+on+vascular+permeability:+an+electron+microscopic+study.">Studies on inflammation. 1. The effect of histamine and serotonin on vascular permeability: an electron microscopic study.</a> J. Biophys. Biochem. Cytol. 11, 571-605 (1961).</li><li>Majno, G., Palade, G. E., Schoefl, G. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+inflammation.+II.+The+site+of+action+of+histamine+and+serotonin+along+the+vascular+tree:+a+topographic+study.">Studies on inflammation. II. The site of action of histamine and serotonin along the vascular tree: a topographic study.</a> J. Biophys. Biochem. Cytol. 11, 607-626 (1961).</li><li>Grant, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+Observation+of+Skeletal+Muscle+Blood+Vessels+(Rat+Cremaster).">Direct Observation of Skeletal Muscle Blood Vessels (Rat Cremaster).</a> J. Physiol. 172, 123-137 (1964).</li><li>Baez, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+open+cremaster+muscle+preparation+for+the+study+of+blood+vessels+by+in+vivo+microscopy.">An open cremaster muscle preparation for the study of blood vessels by in vivo microscopy.</a> Microvasc. Res. 5, 384-394 (1973).</li><li>Bohlen, H. G., Gore, R. W., Hutchins, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+microvascular+pressures+in+normal+and+spontaneously+hypertensive+rats.">Comparison of microvascular pressures in normal and spontaneously hypertensive rats.</a> Microvasc. Res. 13, 125-130 (1977).</li><li>Klitzman, B., Duling, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microvascular+hematocrit+and+red+cell+flow+in+resting+and+contracting+striated+muscle.">Microvascular hematocrit and red cell flow in resting and contracting striated muscle.</a> Am. J. Physiol. 237, 481-490 (1979).</li><li>Hungerford, J. E., Sessa, W. C., &amp;amp, S. e. g. a. l., S, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vasomotor+control+in+arterioles+of+the+mouse+cremaster+muscle.">Vasomotor control in arterioles of the mouse cremaster muscle.</a> FASEB J. 14, 197-207 (2000).</li><li>Figueroa, X. F., Paul, D. L., Simon, A. M., Goodenough, D. A., Day, K. H., Damon, D. N., Duling, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+role+of+connexin40+in+the+propagation+of+electrically+activated+vasodilation+in+mouse+cremasteric+arterioles+in+vivo.">Central role of connexin40 in the propagation of electrically activated vasodilation in mouse cremasteric arterioles in vivo.</a> Circ. Res. 92, 793-800 (2003).</li><li>Wolfle, S. E., Schmidt, V. J., Hoepfl, B., Gebert, A., Alcolea, S., Gros, D., de Wit, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Connexin45+cannot+replace+the+function+of+connexin40+in+conducting+endothelium-dependent+dilations+along+arterioles.">Connexin45 cannot replace the function of connexin40 in conducting endothelium-dependent dilations along arterioles.</a> Circ. Res. 101, 292-1299 (2007).</li><li>Milkau, M., Kohler, R., de Wit, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crucial+importance+of+the+endothelial+K++channel+SK3+and+connexin40+in+arteriolar+dilations+during+skeletal+muscle+contraction.">Crucial importance of the endothelial K+ channel SK3 and connexin40 in arteriolar dilations during skeletal muscle contraction.</a> FASEB J. 24, 3572-3579 (2010).</li><li>Bagher, P., Duan, D., Segal, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+impaired+neurovascular+transmission+in+a+murine+model+of+Duchenne+Muscular+Dystrophy.">Evidence for impaired neurovascular transmission in a murine model of Duchenne Muscular Dystrophy.</a> J. Appl. Physiol. 110, 601-610 (2011).</li><li>Tallini, Y. N., Brekke, J. F., Shui, B., Doran, R., Hwang, S. M., Nakai, J., Salama, G., Segal, S. S., Kotlikoff, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Propagated+endothelial+Ca2++waves+and+arteriolar+dilation+in+vivo:+measurements+in+Cx40BAC-GCaMP2+transgenic+mice.">Propagated endothelial Ca2+ waves and arteriolar dilation in vivo: measurements in Cx40BAC-GCaMP2 transgenic mice.</a> Circ. Res. 101, 1300-1309 (2007).</li><li>Grant, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+denervation+on+skeletal+muscle+blood+vessels+(rat+cremaster).">The effects of denervation on skeletal muscle blood vessels (rat cremaster).</a> J. Anat. 100, 305-316 (1966).</li><li>Proctor, K. G., Busija, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationships+among+arteriolar,+regional,+and+whole+organ+blood+flow+in+cremaster+muscle.">Relationships among arteriolar, regional, and whole organ blood flow in cremaster muscle.</a> Am. J. Physiol. 249, H34-H41 (1985).</li><li>Bagher, P., Segal, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+blood+flow+in+the+microcirculation:+Role+of+conducted+vasodilation.">Regulation of blood flow in the microcirculation: Role of conducted vasodilation.</a> Acta Physiol. , (2011).</li><li>Hill, M. A., Simpson, B. E., Meininger, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+cremaster+muscle+hemodynamics+due+to+disruption+of+the+deferential+feed+vessels.">Altered cremaster muscle hemodynamics due to disruption of the deferential feed vessels.</a> Microvasc. Res. 39, 349-363 (1990).</li></ol>

تمت الموافقة على كافة الإجراءات والبروتوكولات التي تنطوي على الحيوانات من قبل لجنة رعاية الحيوان ، واستخدم من جامعة ميسوري والتي أجريت في الاتفاق مع المرشد لمعاهد الصحة الوطنية لرعاية واستخدام الحيوانات المختبرية. وقد رعت الإنتاج من هذه المادة عن طريق الفيديو بواسطة الضوئيات ستانفورد.

نحن هنا وصف العضلة المشمرة فتح في إعداد الماوس لرصد دوران الأوعية الدقيقة في الجسم الحي. وعلى غرار هذا الإجراء بعد &quot;فتح&quot; إعداد المشمرة وصف لأول مرة في الفئران 4. ويمكن أن يكتمل مع الممارسة العملية الجراحية بالكامل في أقل من 1 ساعة. وبراعة هذا الإعداد يسمح لمجموعة متنوعة من المعالجات التجريبية وتكييفها بسهولة للهامستر وكذلك الفئران ، وتمكين مجموعة متنوعة من نماذج تجريبية لدراستها بطريقة مماثلة. ويقتصر إعداد للحيوانات من الذكور ، وينبغي إجراء قياسات نحو وسط النسيج ، وتجنب المناطق المتضررة بالقرب من حواف العضلات 14. وينبغي أيضا أنه يجب الاعتراف بأنه ، في حين يتم تغيير توزيع الضغط والتدفق عندما يتم قطع الأوعية ربط عضلة المشمرة 16 ، microvessels تظل استجابة ومناسبة لجمع البيانات استنساخه. القيد الرئيسي على الرؤية ودوران الأوعية الدقيقة في العضلات المشمرة هو تراكم النسيج الضام مثل الحيوانات الناضجة وزيادة في الحجم ، ولا سيما في الفئران ولكن أيضا في الهامستر. كذلك ، الحيوانات الحصول على الدهون ، فإنه يصبح من الصعب السيطرة عليها بسبب التخدير بنتوباربيتال محبة للدهون ويمكن استيعابها في الأنسجة الدهنية. أفضل طريقة للمضي قدما هو من خلال التحلي بالصبر مع ضمان الحفاظ على درجة حرارة جسم الحيوان ° ~ في 37 درجة مئوية بينما المروية الأنسجة تتعرض باستمرار مع PSS.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><th> اسم أو جهاز كاشف </th><th> شركة </th><th> فهرس العدد </th><th> تعليقات </th></tr><tr><td> كلوريد الصوديوم </td><td> الصياد </td><td> S642 - 212 </td><td></td></tr><tr><td> كلوريد البوتاسيوم </td><td> سيغما </td><td> P9541 </td><td></td></tr><tr><td> سلفات المغنيزيوم </td><td> سيغما </td><td> M2643 </td><td></td></tr><tr><td> كلوريد الكالسيوم </td><td> سيغما </td><td> C1016 </td><td></td></tr><tr><td> بيكربونات الصوديوم </td><td> الصياد </td><td> S233 </td><td></td></tr><tr><td> Nembutal الصوديوم الحل </td><td> Lundbeck </td><td> NDC 67386-501-55 </td><td> كما يشار إلى الصوديوم بنتوباربيتال </td></tr><tr><td> درجة الحرارة المراقب المالي </td><td> نذير الآلات </td><td> TC - 344B </td><td> البديل : تعديل التيار المستمر 12V العرض </td></tr><tr><td> سلسلة سخان منصة 20 طقم RH - 2 </td><td> نذير الآلات </td><td> 64-0274 </td><td> يتطلب مبنية خصيصا لوحة الألمنيوم </td></tr><tr><td> كاميرا 10 ميجا </td><td> ستانفورد الضوئيات </td><td> XR/Mega-10 </td><td></td></tr><tr><td> MVX10 </td><td> اوليمبوس </td><td></td><td></td></tr><tr><td> MV PLAPO 2XC العدسة </td><td> اوليمبوس </td><td></td><td></td></tr><tr><td> MVX10 الوقوف الهجين </td><td> ليدز </td><td> LBX الهجين </td><td></td></tr><tr><td> مزمر مراقبة البرامج </td><td> ستانفورد الضوئيات </td><td></td><td> برنامج للكاميرا 10 ميجا </td></tr><tr><td> مجهر ستيريو </td><td> نيكون </td><td> SMZ645 </td><td></td></tr><tr><td> سيليكون مانع التسرب للماء (واضح) </td><td> جنرال الكتريك </td><td> 47970-72643 - LW5000 </td><td> مانعات التسرب سيليكون أخرى واضحة تعمل أيضا </td></tr><tr><td> 60 × طبق بيتري 15mm </td><td> الصياد </td><td> 08-757 - 13A </td><td></td></tr><tr><td> Sylgard </td><td> داو كورننغ </td><td> 184 </td><td></td></tr><tr><td> Microdissection مقص </td><td> أدوات العلوم غرامة </td><td> 15003-08 </td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> دومون الملقط </td><td> أدوات العلوم غرامة </td><td> # 5 / 45 </td><td></td></tr><tr><td> GP ميليبور اكسبريس PLUS الغشائية </td><td> ميليبور </td><td> SCGPT05RE </td><td></td></tr><tr><td> دبابيس Minutiens الحشرات </td><td> أوسترليتز </td><td> M حجم 0،15 ملم </td><td></td></tr><tr><td> المضغوط الحيوانات الأليفة المتقلب </td><td> فاهل شركة كليبر </td><td> نموذج 9966 </td><td> نظيفة بعد كل استعمال </td></tr></tbody></table>

the mouse cremaster muscle preparation for intravital imaging of the microcirculation

في جميع أنحاء الجسم ، والحفاظ على التوازن يتطلب امدادات ثابتة من الأوكسجين والمواد المغذية يصاحب ذلك مع إزالة الأيضية والمنتجات. ويتحقق هذا التوازن عن طريق حركة الدم من خلال دوران الأوعية الدقيقة ، والذي يشمل فروع أصغر من إمدادات الأوعية الدموية في جميع الأنسجة والأعضاء. الشرينات فرع من الشرايين على شكل شبكات للتحكم في توزيع وحجم الدم المؤكسج تصب في العديد من الشعيرات الدموية المرتبطة ارتباطا وثيقا مع خلايا متنية. الشعيرات الدموية توفر مساحة كبيرة لتبادل diffusional بين خلايا الأنسجة والدم. الوريدات جمع النفايات السائلة الشعرية وتتلاقى مع عودتهم امؤكسج الدم نحو القلب. لمراقبة هذه العمليات في الوقت الحقيقي يتطلب نهجا تجريبيا لتصور والتلاعب دوران الأوعية الدقيقة الحية. وقد استخدم لأول مرة في عضلة المشمرة الجرذان كنموذج لدراسة التهاب الأنسجة باستخدام المجهر والإلكترون 1،2 آخر بعد الوفاة. التحقيق الأول في تقرير المجراة من الفئران المعرضة سليمة العضلات المشمرة ردود الاوعية الدموية الدقيقة على الأدوية فعال في الأوعية باستخدام الضوء ينعكس 3. مهما كانت محدودة انحناء في العضلات وعدم وجود إنارة ركزت على فائدة من هذا الإعداد. واستتبع فتح فتحا كبيرا في العضلات ، وفصلها من الخصية وانتشاره شعاعيا كورقة للشقة تضوء تحت المجهر المركب 4. في حين أظهرت أن يكون إعداد قيمة لدراسة علم وظائف الأعضاء للدوران الأوعية الدقيقة في الجرذان والفئران البيضاء 5 6 ، وقد ثبت في العضلة المشمرة الفئران 7 مفيدة بشكل خاص في تشريح مسارات الخلوية تشارك في تنظيم وظيفة الاوعية الدموية الدقيقة 11/08 في الوقت الحقيقي والتصوير من بين الخلايا إشارات 12. مشتق من العضلة المشمرة عضلات البطن الداخلية المائل والعرضي والخصيتين ينزل من خلال القناة الإربية 13. إنه يساعد على دعم (باليونانية : المشمرة = رباط الجورب) والحفاظ على درجة حرارة الخصيتين. كما هو موضح هنا ، هو إعداد ورقة والعضلات المشمرة شقة رقيقة لقرار الضوئية المعلقة. مع الحفاظ على الماوس في درجة حرارة الجسم ثابتة وطائرة من التخدير ، وإعداد الجراحية ينطوي على تحرير العضلات من الأنسجة المحيطة بها ، والخصيتين ، ونشر ذلك على قاعدة التمثال من المطاط silastic شفافة وتأمين الحواف مع دبابيس الحشرات بينما لري بشكل مستمر مع محلول ملحي الفسيولوجية . ويستخدم هذا البروتوكول الفئران المعدلة وراثيا في التعبير عن GCaMP2 الخلايا البطانية تصلب. GCaMP2 المرمزة وراثيا هو مؤشر الكالسيوم الفلورسنت 12 الجزيء. Widefield التصوير والكاميرا كثف الجهاز المسؤول إلى جانب تمكين في الدراسة المجراة من إشارات الكالسيوم في بطانة تصلب.

Watch this Scientific Journal Video about The Mouse Cremaster Muscle Preparation for Intravital Imaging of the Microcirculation at JoVE.com

The Mouse Cremaster Muscle Preparation for Intravital Imaging of the Microcirculation

ووصف إعداد التصور والأنسجة لمعالجة التجريبية للدوران الأوعية الدقيقة الحية. في الفئران الذكور تخدير ، يتم إعداد رقيقة ، والعضلات المشمرة أوعية دموية للغاية لintravital المجهري لدراسة شبكات الاوعية الدموية الدقيقة بما في ذلك الشرايين والشعيرات الدموية والأوردة. ويتم تكييف بسهولة التحضير لهذه الجرذان والفئران البيضاء.

العضلة المشمرة ماوس التحضير لتصوير Intravital للدوران الأوعية الدقيقة

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

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32.1K Views.  University of Missouri. ووصف إعداد التصور والأنسجة لمعالجة التجريبية للدوران الأوعية الدقيقة الحية. في الفئران الذكور تخدير ، يتم إعداد رقيقة ، والعضلات المشمرة أوعية دموية للغاية لintravital المجهري لدراسة شبكات الاوعية الدموية الدقيقة بما في ذلك الشرايين والشعيرات الدموية والأوردة. وي...

Video: The Mouse Cremaster Muscle Preparation for Intravital Imaging of the Microcirculation

Microiontophoresis and Micromanipulation for Intravital Fluorescence Imaging of the Microcirculation

Microiontophoresis entails passage of current through a micropipette tip to deliver a solute at a designated site within an experimental preparation. Microiontophoresis can simulate synaptic transmission1 by delivering neurotransmitters and neuropeptides onto neurons reproducibly2. Negligible volume (fluid) displacement avoids mechanical disturbance to the experimental preparation. Adapting these techniques to the microcirculation3 has enabled mechanisms of vasodilation and vasoconstriction to be studied at the microscopic level in vivo4,5. A key advantage of such localized delivery is enabling vasomotor responses to be studied at defined sites within a microvascular network without evoking systemic or reflexive changes in blood pressure and tissue blood flow, thereby revealing intrinsic properties of microvessels.
A limitation of microiontophoresis is that the precise concentration of agent delivered to the site of interest is difficult to ascertain6. Nevertheless, its release from the micropipette tip is proportional to the intensity and duration of the ejection current2,7, such that reproducible stimulus-response relationships can be readily determined under defined experimental conditions (described below). Additional factors affecting microiontophoretic delivery include solute concentration and its ionization in solution. The internal diameter of the micropipette tip should be &tilde; 1 &mu;m or less to minimize diffusional 'leak', which can be counteracted with a retaining current. Thus an outward (positive) current is used to eject a cation and a negative current used to retain it within the micropipette.
Fabrication of micropipettes is facilitated with sophisticated electronic pullers8. Micropipettes are pulled from glass capillary tubes containing a filament that 'wicks' solution into the tip of the micropipette when filled from the back end ("backfilled"). This is done by inserting a microcapillary tube connected to a syringe containing the solution of interest and ejecting the solution into the lumen of the micropipette. Micromanipulators enable desired placement of micropipettes within the experimental preparation. Micromanipulators mounted on a movable base can be positioned around the preparation according to the topography of microvascular networks (developed below). 
The present protocol demonstrates microiontophoresis of acetylcholine (ACh+ Cl-) onto an arteriole of the mouse cremaster muscle preparation (See associated protocol: JoVE ID#2874) to produce endothelium-dependent vasodilation. Stimulus delivery is synchronized with digitized image acquisition using an electronic trigger. The use of Cx40BAC-GCaMP2 transgenic mice9 enables visualization of intracellular calcium responses underlying vasodilation in arteriolar endothelial cells in the living microcirculation.

Microiontophoresis entails movement of ions from a micropipette in response to a difference in electrical potential between the inside and outside of the micropipette. Biologically active molecules are thereby delivered in proportion to electrical current. We illustrate acetylcholine microiontophoresis in conjunction with micromanipulation to study endothelium-dependent vasodilation in the microcirculation.

Microiontophoresis والمجهرية للتصوير الإسفار Intravital للدوران الأوعية الدقيقة

Microiontophoresis entails passage of current through a micropipette tip to deliver a solute at a designated site within an experimental  preparation. ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

جيل من الفئران المستمدة من الخلايا الجذعية المحفزة المستحثة

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

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.

حيوي داخلي المجهري للدوران الأوعية الدقيقة في العضلات المشمرة ماوس لتحليل الهجرة الجذعية الطرفية المحمولة

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

Isolation of Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High Throughput Microplate Respiratory Measurements

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial respirometric assays from isolated mitochondrial preparations allow for the assessment of mitochondrial function, as well as determination of the mechanism(s) of action of drugs and proteins that modulate metabolism. Current isolation procedures often require large quantities of tissue to yield high quality mitochondria necessary for respirometric assays. The methods presented herein describe how high quality purified mitochondria (~ 450 &#181;g) can be isolated from minimal quantities (~75-100 mg) of mouse skeletal muscle for use in high throughput respiratory measurements. We determined that our isolation method yields 92.5&#177; 2.0% intact mitochondria by measuring citrate synthase activity spectrophotometrically. In addition, Western blot analysis in isolated mitochondria resulted in the faint expression of the cytosolic protein, GAPDH, and the robust expression of the mitochondrial protein, COXIV. The absence of a prominent GAPDH band in the isolated mitochondria is indicative of little contamination from non-mitochondrial sources during the isolation procedure. Most importantly, the measurement of O2 consumption rate with micro-plate based technology and determining the respiratory control ratio (RCR) for coupled respirometric assays shows highly coupled (RCR; &#62;6 for all assays) and functional mitochondria. In conclusion, the addition of a separate mincing step and significantly reducing motor driven homogenization speed of a previously reported method has allowed the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle that results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

Here, we present a modification of a previously reported method that allows for the isolation of high quality and purified mitochondria from smaller quantities of mouse skeletal muscle. This procedure results in highly coupled mitochondria that respire with high function during microplate based respirometirc assays.

عزل الميتوكوندريا من كميات الحد الأدنى من الفأر العضلات الهيكلية للإنتاجية عالية صفيحة ميكروسكوبية الجهاز التنفسي القياسات

Dysfunctional skeletal muscle mitochondria play a role in altered metabolism observed with aging, obesity and Type II diabetes. Mitochondrial ...

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.

عزل الفئران التاجي الأوعية الدموية خلايا العضلات الملساء

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

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.

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.

التصور النوعي والكمي للطبقة خالية من الخلايا في الشرايين من العضلات الفئران المشمرة

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 ...

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.

بروتوكول الآلي السريع لالعضلات تحليل الألياف السكان في الجرذ العضلات المقاطع العرضية عن طريق سلسلة الميوسين الثقيلة المناعية

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

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is regulated spatially and temporally. The use of a fluorescent gene under the control of an immunity-related promoter in combination with an automated fluorescence microscopy is a simple way to understand spatiotemporal regulation of plant immunity. In contrast to the root tissues that have been used for a number of various intravital fluorescent imaging experiments, there exist few fluorescent live-imaging examples for the leaf tissues that encounter an array of airborne microbial infections. Therefore, we developed a simple method to mount leaves of Arabidopsis thaliana plants for live-cell imaging over an extended period of time. We used transgenic Arabidopsis plants expressing the yellow fluorescent protein (YFP) genefused to the nuclear localization signal (NLS) under the control of the promoter of a defense-related marker gene, Pathogenesis-Related 1 (PR1). We infiltrated a transgenic leaf with Pseudomonas syringae pv. tomato DC3000 (avrRpt2) strain (Pst_a2) and performed in vivo time-lapse imaging of the YFP signal for a total of 40 h using an automated fluorescence stereomicroscope. This method can be utilized not only for studies on plant immune responses but also for analyses of various developmental events and environmental responses occurring in leaf tissues.

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

We report a simple and versatile method for performing fluorescent live-imaging of Arabidopsis thaliana leaves over an extended period of time. We use a transgenic Arabidopsis plant expressing a fluorescent reporter gene under the control of an immunity-related promoter as an example for gaining spatiotemporal understanding of plant immune responses.

أسلوب متعدد الاستخدامات لتركيب نبات يترك لتصوير الوقت الفاصل بين إينترافيتال

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is ...

Sample Preparation for Metabolic Profiling using MALDI Mass Spectrometry Imaging

Metabolomics, the study to identify and quantify small molecules and metabolites present in an experimental sample, has emerged as an important tool to investigate the biological activities during development and diseases. Metabolomics approaches&#160;are widely employed in the study of cancer, nutrition/diet, diabetes, and other physiological and pathological conditions involving metabolic processes. An advantageous tool that aids in metabolomic profiling advocated in this paper is matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI). Its ability to detect metabolites in situ without labeling, structural modifications, or other specialized reagents, such as those used in immunostaining, makes MALDI MSI a unique tool in advancing methodologies relevant in the field of metabolomics. An appropriate sample preparation process is critical to yield optimal results and will be the focus of this paper.

The goal of this protocol is to provide detailed guidance on the sample preparation when planning for experiments using MALDI MSI to maximize metabolic and molecular detection in biological samples.

إعداد عينة للتنميط الأيضي باستخدام التصوير الطيفي الشامل MALDI

Metabolomics, the study to identify and quantify small molecules and metabolites present in an experimental sample, has emerged as an important tool to ...

An Improved Chemotaxis Assay for the Rapid Identification of Rhizobacterial Chemoattractants in Root Exudates

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward method to quickly identify the chemoattractants that could induce the chemotactic movement of rhizosphere growth-promoting bacteria on sterile glass slides via simple steps. Bacteria solution (OD600 = 0.5) and sterile chemoattractant aqueous solution were added dropwise on the glass slide at an interval of 1 cm. An inoculating loop was used to connect the chemoattractant aqueous solution to the bacterial solution. The slide was kept at room temperature for 20 min on the clean bench. Finally, the chemoattractant aqueous solution was collected for bacterial counting and microscopic observation. In this study, through multiple comparisons of experimental results, the method overcame multiple shortcomings of traditional bacterial chemotaxis methods. The method reduced the error of plate counting and shortened the experimental cycle. For the identification of chemoattractant substances, this new method can save 2-3 days compared to the traditional method. Additionally, this method allows any researcher to systematically complete a bacterial chemotaxis experiment within 1-2 days. The protocol can be considered a valuable resource for understanding plant-microbe interactions.

Here, we present an improved chemotaxis assay protocol. The goal of this protocol is to reduce the steps and costs of traditional bacterial chemotaxis methods and to serve as a valuable resource for understanding plant-microbe interactions.

فحص كيميائي محسن للتعرف السريع على الجاذبات الكيميائية الريزوبكتيرية في إفرازات الجذر

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward ...