وأيد هذه الدراسة في جزء من برنامج الحكومة OIS الفيكتوري ، والوطني للصحة ومجلس البحوث الطبية من برنامج المنح والمشاريع أستراليا (JPF شين الغبار) ، والمنح الدراسية للدراسات العليا (D ميشل).

1. عزل الشرايين السباتية <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> Euthanase10 الاسبوع القديمة سبراغ داولي الفئران عبر CO 2 / O 2 الاختناق. </li><li style=";text-align:right;direction:rtl"> اليمين واليسار الموحد المكوس الشرايين السباتية مع الأبهر والقلب ضمان الحد الأدنى تمتد من السفن. </li><li style=";text-align:right;direction:rtl"> في المخزن الجليد الباردة كريبس فصل الشرايين السباتية من الشريان الأورطي والقلب وأداء تشريح قريبة. </li><li style=";text-align:right;direction:rtl"> تبقي السفن معزولة في كريبس على الجليد قبل التركيب. </li></ol> تقريبا. الساعة = 45 دقيقة 2. فتيلة السفينة الغرفة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> في الموصلات القريبة والبعيدة من غرفة عازلة سفينة كريبس تدفق الحفاظ على درجة الحموضة الفسيولوجية عن طريق غرس كربوجين الغاز (95 ٪ O 2 ، 5 ٪ CO 2) من خلال المنطقة العازلة في 37 درجة مئوية. </li><li style=";text-align:right;direction:rtl"> ضمان أنابيب ومسح cannulas تماما ويتم محاذاة. </li><li style=";text-align:right;direction:rtl"> تدفق كريب العازلة من خلال P1 (القريبة) ومحولات الطاقة P2 (قاص). إغلاق صنابير المياه لضمان عدم وجود فقاعات الهواء في محولات الطاقة. </li><li style=";text-align:right;direction:rtl"> ربط المحولات في موصلات المقابلة مرة أخرى وتدفق أكثر كريبس العازلة ضمان عدم فقاعات الهواء. إغلاق الصنابير الى قاعة المحكمة. </li></ol> تقريبا. الساعة = 15 دقيقة 3. غرفة الضغط على سفينة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> بدوره على معدات الضغط : أجهزة الضغط ومراقبة ضغط ، ومضخة تمعجية (الشكل 1). </li><li style=";text-align:right;direction:rtl"> مع مضخة تمعجية على الضغط والضغط على أجهزة أوتوماتيكية العازلة كريبس من خلال تشغيل الأنبوب عند 20 مم زئبق (الطلب في 1) ، وضمان عدم فقاعات الهواء. </li><li style=";text-align:right;direction:rtl"> توصيل أنابيب للمحول P2 مغلقة. وسوف تصبح ضغط مستقرة. </li><li style=";text-align:right;direction:rtl"> فتح الصنبور P2 إلى غرفة سفينة لطرد أي فقاعات ثم أغلق الخروج. </li><li style=";text-align:right;direction:rtl"> ملء حوض الاستحمام مع العازلة كريبس (5 -- 7 مل). </li></ol> تقريبا. الساعة = 10 دقيقة 4. تصاعد السفينة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تحت المجهر مكان تشريح العلاقات البوليستر الأسود على كل قنية (الشكل 2). </li><li style=";text-align:right;direction:rtl"> نقل cannulas وأصحاب قنية ، وبصرف النظر جبل. ¼ السفينة (قوس الأبهر نهاية) على قنية P1 ضمان عدم تمزق السفينة. </li><li style=";text-align:right;direction:rtl"> استخدام حقنة مليئة العازلة كريبس تدفق الدم الزائد بلطف من السفينة عن طريق محول P1. إغلاق P1 إلى الغرفة. </li><li style=";text-align:right;direction:rtl"> تأمين السفينة إلى قنية P1 مع ربطة عنق البوليستر. </li><li style=";text-align:right;direction:rtl"> نقل cannulas / أصحاب قنية أقرب للتركيب على القنية P2. </li><li style=";text-align:right;direction:rtl"> جبل نهاية البعيدة للسفينة (~ ¼ طول) على القاصي قنية. آمنة مع ربطة عنق البوليستر. </li></ol> تقريبا. الساعة = 20 دقيقة 5. الضغط على سفينة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ضبط أصحاب قنية لضمان عدم الانحناء أو التمدد في السفينة. </li><li style=";text-align:right;direction:rtl"> مع P1 و P2 مغلقة إلى غرفة تبديل أجهزة ضغط من تلقائي إلى يدوي. التحقق من مضاعفات ضغط عدم وجود انخفاض في ضغط متواصل في غضون 10 ثانية. إذا كان هناك ، يتم حدوث تسرب وصلات ومحولات الطاقة تحتاج إلى المضمون في المكان. </li><li style=";text-align:right;direction:rtl"> ضبط مضاعفات ضغط مرة أخرى إلى التلقائي P2 ثم فتح الى قاعة المحكمة. تحت المجهر مراقبة السفينة تمدد عند فتح الصنبور إلى الغرفة. </li><li style=";text-align:right;direction:rtl"> تبديل أجهزة الضغط إلى يدوي. الاختيار مرة أخرى للانخفاض المستمر في الضغط في غضون 10 ثانية. إذا كان هناك تسرب ثم النظام لا ضغط ضيق وهناك من المرجح أن يكون ثقبا في السفينة. </li><li style=";text-align:right;direction:rtl"> التبديل إلى P1 فتح التلقائي للغرفة. على إعداد دليل تأكد من أن الضغوط لا تزال مستقرة. </li><li style=";text-align:right;direction:rtl"> إذا لم يحصل أي تسرب ، على إعداد دليل لزيادة الطلب 2 (40 مم زئبق). </li><li style=";text-align:right;direction:rtl"> التكيف التلقائي ومراقبة السفينة تحت المجهر. إذا لزم الأمر ، وضبط صاحب قنية لضمان عدم خضوع في vessel.Check عن التسريبات حول إعداد يدوي. </li><li style=";text-align:right;direction:rtl"> كرر الخطوات من 5،6-5،7 زيادة الطلب على 3 (60 ملم زئبقي) ، 4 (80 مم زئبق) وهلم جرا ، حتى يتم التوصل إلى الضغط المطلوب. </li></ol> تقريبا. الوقت = 15 -- 30 دقيقة 6. تفرخ السفينة الضغط <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> مجموعة الاتصال وحدة تحكم في درجة الحرارة إلى 37 درجة مئوية. </li><li style=";text-align:right;direction:rtl"> مع الثانية مضخة تمعجية العازلة كريبس يروي (1 مل / دقيقة) في الإناء حمام الغرفة. </li><li style=";text-align:right;direction:rtl"> الاتصال تتم إزالة الشافطة لضمان الغرفة فقط الطبقة العليا من حوض الاستحمام. </li><li style=";text-align:right;direction:rtl"> احتضان سفينة الضغط على 37 درجة مئوية لمدة 1 ساعة مع الضغط لتعيين التلقائي. </li><li style=";text-align:right;direction:rtl"> تحقق من عدم حدوث أي تسرب الضغط (التحول إلى دليل) بشكل دوري. </li><li style=";text-align:right;direction:rtl"> ويمكن ملاحظة آثار التدخلات الدوائية المختلفة ببساطة عن طريق إضافة إلى مجمع الحمام أثناء الحضانة. </li></ol> تقريبا. الوقت = 1 ساعة 7. Perfusing السفينة مع ضغط الدم الكامل <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> خلال الحصول على حضانة على الأقل 7.5 مل من الدم البشري كله / السفينة التي تم جمعها في الهيبارين يو 40 / مليلتر دم. في احتضان الصقر 50 مل عند 37 درجة مئوية. </li><li style=";text-align:right;direction:rtl"> 10 دقائق قبل نهاية طنانه الدم حضانة التسمية مع VybrantDil (1:1000) لمدة 10 دقيقة عند 37 درجة مئوية في الظلام. </li><li style=";text-align:right;direction:rtl"> بعد 10 دقيقة ، وجمع الدم في حقنة إزالة أي فقاعات ، ونعلق على مضخة المحاقن مع سترة الحرارة وضعت في 37 درجة مئوية. </li><li style=";text-align:right;direction:rtl"> إغلاق محول P1 للغرفة / السفينة والمحاقن وتوصيل أنابيب النفايات. </li><li style=";text-align:right;direction:rtl"> تطهير الدم في 1000 ميكرولتر / دقيقة عن طريق أنابيب النفايات لإزالة أي فقاعات. </li><li style=";text-align:right;direction:rtl"> فتح P1 الى قاعة المحكمة. فإن أجهزة ضبط الضغط تلقائيا للحفاظ على الضغط المطلوب. </li><li style=";text-align:right;direction:rtl"> يروي الدم عند 100 ميكرولتر / دقيقة. </li><li style=";text-align:right;direction:rtl"> باستخدام المجهر الفلورسنت بالإضافة إلى كاميرا رقمية حقول two سجل السفينة perfused لمدة 15 ثانية في 1 و 3 و 5 و 7.5 و 10 دقائق. </li><li style=";text-align:right;direction:rtl"> ويمكن تقييم سلامة نضح آخر وظيفة بطانة الأوعية الدموية عن طريق تقنيات مثل الدوائية يمكن تحديد myograph والتصاق جزيء التعبير التي المناعية للتحقق من صحة مزيد من الاستجابة للالتهابات. </li></ol> تقريبا. الساعة = 15 دقيقة 8. ممثل النتائج : ويظهر رسم تخطيطي للضغط الإعداد الغرفة في الشكل 1. ويمكن تصور أن يكون مع كاميرا رقمية بالإضافة إلى النتائج المجهر الفلورسنت على الفور عن طريق تسجيلات فيديو حية. وينظر الى صور الفيديو ممثل في الشكل 3 حيث تعتبر الكريات البيضاء لتكون ملتصقة على البطانة اذا ظلت ثابتة لمدة 10 ثانية. يمكن مع تسجيلات فيديو على الخلايا حلقة مستمرة انضمت تحسب حقل متوسط. في حين أن كلا منخفضة (الشكل 3A) وعالية (الشكل 3B) الضغط سوف يسبب قدرا من الالتصاق وينظر الى زيادة كبيرة في التصاق الكريات البيض في الضغط العالي داخل اللمعة ويتجلى هذا أيضا كميا (الشكل 4). <img alt="الشكل 1" src="/files/ftp_upload/3221/3221fig1.jpg" /> الشكل 1. مضغوطة سفينة فيفو السابقين التخطيطي الغرفة. سفينة مقنى متصلا محول (P1) القريبة والبعيدة (P2) محولات ضغط الدم الذي يمكن أن يتم التلاعب داخل السفينة. يتم الاحتفاظ نضح من خلال محول P1 و P2 الضغط عبر محول. <img alt="الشكل 2" src="/files/ftp_upload/3221/3221fig2.jpg" /> الشكل 2. Cannuala مع العلاقات بين البلدين. مرفقة العلاقات البوليستر الأسود إلى كل قنية. <img alt="الشكل 3" src="/files/ftp_upload/3221/3221fig3.jpg" /> الشكل 3. صور الفيديو التمثيلية. الحيوي التصاق الخلية (السهم الأحمر) تحت مضان في 80 مم زئبق (أ) و 120 مم زئبق (B) بعد 10 دقيقة من نضح. <img alt="الشكل 4" src="/files/ftp_upload/3221/3221fig4.jpg" /> الشكل 4. السباتي التصاق كريات بيضاء في الشرايين بعد سبراغ داولي حضانة ساعة 1 في انخفاض (80 مم زئبقي) والضغط العالي (120 مم زئبق). *** P &lt;0.001 ، وتحليلها وبنسبة 2 في اتجاه والتدابير المتكررة ANOVA باستخدام اختبار آخر مخصص Bonferroni.

<ol>
	<li>Lopez, A. D., Mathers, C. D., Ezzati, M., Jamison, D. T., Murray, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+and+regional+burden+of+disease+and+risk+factors.">Global and regional burden of disease and risk factors.</a> Lancet. 367, 1747-1757 (2001).</li><li>Szczech, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+kidney+injury+and+cardiovascular+outcomes+in+acute+severe+hypertension.">Acute kidney injury and cardiovascular outcomes in acute severe hypertension.</a> Circulation. 121, 2183-2191 .</li><li>Rodriguez-Garcia, J. L., Botia, E., de La Sierra, A., Villanueva, M. A., Gonzalez-Spinola, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Significance+of+elevated+blood+pressure+and+its+management+on+the+short-term+outcome+of+patients+with+acute+ischemic+stroke.">Significance of elevated blood pressure and its management on the short-term outcome of patients with acute ischemic stroke.</a> Am J Hypertens. 18, 379-384 (2005).</li><li>Levy, D., Larson, M. G., Vasan, R. S., Kannel, W. B., Ho, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+progression+from+hypertension+to+congestive+heart+failure.">The progression from hypertension to congestive heart failure.</a> JAMA. 275, 1557-1562 (1996).</li><li>Ley, K., Laudanna, C., Cybulsky, M. I., Nourshargh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Getting+to+the+site+of+inflammation:+the+leukocyte+adhesion+cascade+updated.">Getting to the site of inflammation: the leukocyte adhesion cascade updated.</a> Nat Rev Immunol. 7, 678-689 (2007).</li><li>Riou, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+pressure+promotes+monocyte+adhesion+to+the+vascular+wall.">High pressure promotes monocyte adhesion to the vascular wall.</a> Circ Res. 100, 1226-1233 (2007).</li><li>Ley, K., Gaehtgens, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial,+not+hemodynamic,+differences+are+responsible+for+preferential+leukocyte+rolling+in+rat+mesenteric+venules.+Circ+Res.">Endothelial, not hemodynamic, differences are responsible for preferential leukocyte rolling in rat mesenteric venules. Circ Res.</a> 69, 1034-1041 (1991).</li><li>Bernhagen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MIF+is+a+noncognate+ligand+of+CXC+chemokine+receptors+in+inflammatory+and+atherogenic+cell+recruitment.">MIF is a noncognate ligand of CXC chemokine receptors in inflammatory and atherogenic cell recruitment.</a> Nat Med. 13, 587-596 (2007).</li><li>Woollard, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiological+levels+of+soluble+P-selectin+mediate+adhesion+of+leukocytes+to+the+endothelium+through+Mac-1+activation.">Pathophysiological levels of soluble P-selectin mediate adhesion of leukocytes to the endothelium through Mac-1 activation.</a> Circ Res. 103, 1128-1138 (2008).</li><li>Eberth, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Importance+of+pulsatility+in+hypertensive+carotid+artery+growth+and+remodeling.">Importance of pulsatility in hypertensive carotid artery growth and remodeling.</a> J Hypertens. 27, 2010-2021 (2009).</li><li>Cooke, J. P., Rossitch, E., Andon, N. A., Loscalzo, J., Dzau, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+activates+an+endothelial+potassium+channel+to+release+an+endogenous+nitrovasodilator.">Flow activates an endothelial potassium channel to release an endogenous nitrovasodilator.</a> J Clin Invest. 88, 1663-1671 (1991).</li></ol>

تمت الموافقة على بروتوكول الدراسة من البحوث الطبية والتعليم ألفريد المخفر الحيوان جنة الأخلاقيات ومستشفى ألفريد جنة الأخلاقيات.

هذا هو أسلوب لدراسة تعديل لالتصاق الكريات البيض في بطانة الأوعية الدموية سليمة في ظل ظروف الضغط معزولة في الوقت الحقيقي. نضح من الغرفة وحدها السفينة تمكن من التحقق من صحة السريع الموالية للالتهابات سلالات من الفئران الكبيرة والسفن الفئران. تمكين التلاعب الضغط يسمح لوحظ من التفاعلات الحيوية الخلية المنخفض لضغوط داخل اللمعة عالية جدا ، وبالتالي أفضل تقليد جي الظروف الفسيولوجية والمرضية في جسم المريض. ويمكن أيضا للسفن القطر يقاس باستخدام خلايا كافية تصوير برنامج ويمكن تحديد تدفق القص وبالتالي معدل وبالتالي التلاعب. مع قدرات myograph والخمسين ، التدخلات الدوائية وضعها في حمام تضيف بعدا آخر للظروف تجريبية محتملة مع هذا النموذج من الدراسات تمكين المسارات الآلية والإشارات. في حين لا يستطيع المحافظة على البطانية خلال التلاعب وأكد أن الضغوط ، يمكن أن تتم الاستجابة لأدن تشافيز وPE نضح آخر 9. وتجدر الإشارة إلى أن هذا يدل على الإعداد آثار الضغط داخل اللمعة على الخلية الى خلية التفاعلات لا آثار تدفق الدم الانقباضي أو نابض ولا الضغوط الانبساطي. وعلاوة على ذلك ، في حين لوحظت تغييرات الضغط الحاد على التصاق الكريات البيض ، ويمكن استخدام هذا الإعداد أيضا أن ننظر إلى آثار الضغط المزمن (أي زيادة مرات حضانة واستخدام الحيوانات الضغوط المزمنة نموذجا). وأظهرت سبراغ الشرايين السباتية داولي المشتركة في هذه المجموعة لكنها يمكن أن تستخدم السلالات والأنواع الأخرى مع إجراء التعديلات المناسبة لحجم قنية. في الواقع ، فمن المهم أن نلاحظ أن العمر والوزن من الحيوانات تؤثر على حجم السفينة ، وبالتالي أن مجموعة يحتاج إلى ما يقارب تكون فردية عن كل سفينة. يمكن تشريح وثيق ودقيق لتحسين التصور النسيج الضام من الكريات البيض كثيرا.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><th> اسم كاشف / المعدات </th><th> شركة </th><th> فهرس العدد </th><th> تعليقات </th></tr><tr><td> مجهر </td><td> كارل زايس ، وشركة </td><td> STEREO ديسكفري V.20 </td><td></td></tr><tr><td> الدكتوراه مضخة الحقنة 2000 </td><td> جهاز هارفارد </td><td> 70-2016 </td><td></td></tr><tr><td> كاميرا رقمية والمراقب المالي </td><td> هاماماتسو ORCA - ER </td><td> C4742 - 95 </td><td></td></tr><tr><td> مضان نظام الإضاءة. </td><td> التجويف حيوية </td><td> X - سايت 120 </td><td></td></tr><tr><td> سفينة غرفة </td><td> نظم التهوية المعيشة </td><td> CH/1/SH </td><td></td></tr><tr><td> الضغط ومضخة هيدروليك المراقب تحوي </td><td> نظم التهوية المعيشة </td><td> PS -- 200 </td><td></td></tr><tr><td> ضغط التروية مراقب </td><td> نظم التهوية المعيشة </td><td> م -- 4 </td><td></td></tr><tr><td> 2 X محول الضغط </td><td> نظم التهوية المعيشة </td><td> PT -- F </td><td></td></tr><tr><td> درجة الحرارة المراقب المالي </td><td> نظم التهوية المعيشة </td><td> TC - 01 </td><td></td></tr><tr><td> مضخة تمعجية </td><td> Instech المختبرات ، وشركة </td><td> P720 </td><td></td></tr><tr><td> VybrantDil الخلية وضع العلامات الحل </td><td> Invitrogen </td><td> V - 22885 </td><td> استخدام 1:1000 </td></tr></tbody></table>

imaging leukocyte adhesion to the vascular endothelium at high intraluminal pressure

Worldwide, hypertension is reported to be in approximately a quarter of the population and is the leading biomedical risk factor for mortality worldwide. In the vasculature hypertension is associated with endothelial dysfunction and increased inflammation leading to atherosclerosis and various disease states such as chronic kidney disease2, stroke3 and heart failure4. An initial step in vascular inflammation leading to atherogenesis is the adhesion cascade which involves the rolling, tethering, adherence and subsequent transmigration of leukocytes through the endothelium. Recruitment and accumulation of leukocytes to the endothelium is mediated by an upregulation of adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), intracellular cell adhesion molecule-1 (ICAM-1) and E-selectin as well as increases in cytokine and chemokine release and an upregulation of reactive oxygen species5. In vitro methods such as static adhesion assays help to determine mechanisms involved in cell-to-cell adhesion as well as the analysis of cell adhesion molecules. Methods employed in previous in vitro studies have demonstrated that acute increases in pressure on the endothelium can lead to monocyte adhesion, an upregulation of adhesion molecules and inflammatory markers6 however, similar to many in vitro assays, these findings have not been performed in real time under physiological flow conditions, nor with whole blood. Therefore, in vivo assays are increasingly utilised in animal models to demonstrate vascular inflammation and plaque development. Intravital microscopy is now widely used to assess leukocyte adhesion, rolling, migration and transmigration7-9. When combining the effects of pressure on leukocyte to endothelial adhesion the in vivo studies are less extensive. One such study examines the real time effects of flow and shear on arterial growth and remodelling but inflammatory markers were only assessed via immunohistochemistry10. Here we present a model for recording leukocyte adhesion in real time in intact pressurised blood vessels using whole blood perfusion. The methodology is a modification of an ex vivo vessel chamber perfusion model9 which enables real-time analysis of leukocyte -endothelial adhesive interactions in intact vessels. Our modification enables the manipulation of the intraluminal pressure up to 200 mmHg allowing for study not only under physiological flow conditions but also pressure conditions. While pressure myography systems have been previously demonstrated to observe vessel wall and lumen diameter11 as well as vessel contraction this is the first time demonstrating leukocyte-endothelial interactions in real time. Here we demonstrate the technique using carotid arteries harvested from rats and cannulated to a custom-made flow chamber coupled to a fluorescent microscope. The vessel chamber is equipped with a large bottom coverglass allowing a large diameter objective lens with short working distance to image the vessel. Furthermore, selected agonist and/or antagonists can be utilized to further investigate the mechanisms controlling cell adhesion. Advantages of this method over intravital microscopy include no involvement of invasive surgery and therefore a higher throughput can be obtained. This method also enables the use of localised inhibitor treatment to the desired vessel whereas intravital only enables systemic inhibitor treatment.

Watch this Scientific Journal Video about Imaging Leukocyte Adhesion to the Vascular Endothelium at High Intraluminal Pressure at JoVE.com

Imaging Leukocyte Adhesion to the Vascular Endothelium at High Intraluminal Pressure

هذا هو أسلوب لتصور التصاق الكريات البيض في بطانة الأوعية في الضغط المقطوع. هذه التقنية تتيح دراسة التصاق الأوعية الدموية تحت تدفق القص مع اختلاف الضغوط داخل اللمعة ما يصل الى 200 ملم زئبقي وبالتالي تقليد جي الظروف المرضية في جسم المريض من ارتفاع ضغط الدم.

التصوير التصاق كريات بيضاء لبطانة الأوعية الدموية في الضغط داخل اللمعة السامية

Worldwide, hypertension is reported to be in approximately a quarter of the population and is the leading biomedical risk factor for mortality worldwide. ...

imaging-leukocyte-adhesion-to-vascular-endothelium-at-high

Research

JoVE Journal

Immunology and Infection

15.6K Views.  Monash University. هذا هو أسلوب لتصور التصاق الكريات البيض في بطانة الأوعية في الضغط المقطوع. هذه التقنية تتيح دراسة التصاق الأوعية الدموية تحت تدفق القص مع اختلاف الضغوط داخل اللمعة ما يصل الى 200 ملم زئبقي وبالتالي تقليد جي الظروف المرضية في جسم المريض من ارتفاع ضغط الدم.

Video: Imaging Leukocyte Adhesion to the Vascular Endothelium at High Intraluminal Pressure

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists. The value of 2P microscopy is that it provides rich spatiotemporal information regarding cell behaviors within intact tissues and in live mice. Leukocyte recruitment plays a significant role in host defense against infection and when unchecked, can contribute to inflammatory and autoimmune diseases. Studying leukocyte recruitment in vivo is technically challenging since cells are moving rapidly within vessels located deep within light scattering tissues. To date, most intravital imaging studies require surgical preparation to expose the blood vessels and tissues. To avoid the tissue damage and inflammation induced by surgery itself, here, we describe a non-invasive single-cell imaging approach that can be used to study leukocyte trafficking in the mouse footpad and phalanges. We discuss the technical aspects of our 2P imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

Non-invasive Imaging of Leukocyte Homing and Migration in vivo

Here, we describe a non-invasive two-photon (2P) microscopy approach to study leukocyte homing in the mouse footpad. We discuss the technical aspects of our tissue imaging preparation and walk the reader through a typical experiment from initial set up to execution and data collection.

غير الغازية تصوير الزاجل كريات بيضاء والهجرة في الجسم الحي</em

Two-photon (2P) microscopy is a high resolution imaging technique that has been broadly adapted by biologists.  The value of 2P microscopy is that it ...

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of guard cells, which are able to close the stomatal pore in response to a number of external factors including light intensity, carbon dioxide concentration, and relative humidity (RH). The stomatal pore is also the main route for pathogen entry into leaves, a crucial step for disease development. Recent studies have unveiled that closure of the pore is effective in minimizing bacterial disease development in Arabidopsis plants; an integral part of plant innate immunity. Previously, we have used epidermal peels to assess stomatal response to live bacteria (Melotto et al. 2006); however maintaining favorable environmental conditions for both plant epidermal peels and bacterial cells has been challenging. Leaf epidermis can be kept alive and healthy with MES buffer (10 mM KCl, 25 mM MES-KOH, pH 6.15) for electrophysiological experiments of guard cells. However, this buffer is not appropriate for obtaining bacterial suspension. On the other hand, bacterial cells can be kept alive in water which is not proper to maintain epidermal peels for long period of times. When an epidermal peel floats on water, the cells in the peel that are exposed to air dry within 4 hours limiting the timing to conduct the experiment. An ideal method for assessing the effect of a particular stimulus on guard cells should present minimal interference to stomatal physiology and to the natural environment of the plant as much as possible. We, therefore, developed a new method to assess stomatal response to live bacteria in which leaf wounding and manipulation is greatly minimized aiming to provide an easily reproducible and reliable stomatal assay. The protocol is based on staining of intact leaf with propidium iodide (PI), incubation of staining leaf with bacterial suspension, and observation of leaves under laser scanning confocal microscope. Finally, this method allows for the observation of the same live leaf sample over extended periods of time using conditions that closely mimic the natural conditions under which plants are attacked by pathogens.

We have developed a simple and reproducible protocol to access stomatal response to live bacteria. This method minimizes wounding and manipulation of the leaf as compared to the use of epidermal peels reported previously.

تقييم الاستجابة للعيش فوهي الخلايا البكتيرية باستخدام التصوير الجامعة ليف

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

إدخال إجهاد القص في دراسة الالتصاق الجرثومي

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Real-time Imaging of Heterotypic Platelet-neutrophil Interactions on the Activated Endothelium During Vascular Inflammation and Thrombus Formation in Live Mice

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 During thrombus formation at the site of arteriolar injury, platelets adherent to the activated endothelium and subendothelial matrix proteins support neutrophil rolling and adhesion.3 Conversely, under venular inflammatory conditions, neutrophils adherent to the activated endothelium can support adhesion and accumulation of circulating platelets. Heterotypic platelet-neutrophil aggregation requires sequential processes by the specific receptor-counter receptor interactions between cells.4 It is known that activated endothelial cells release adhesion molecules such as von Willebrand factor, thereby initiating platelet adhesion and accumulation under high shear conditions.5 Also, activated endothelial cells support neutrophil rolling and adhesion by expressing selectins and intercellular adhesion molecule-1 (ICAM-1), respectively, under low shear conditions.4 Platelet P-selectin interacts with neutrophils through P-selectin glycoprotein ligand-1 (PSGL-1), thereby inducing activation of neutrophil &beta;2 integrins and firm adhesion between two cell types. Despite the advances in in vitro experiments in which heterotypic platelet-neutrophil interactions are determined in whole blood or isolated cells,6,7 those studies cannot manipulate oxidant stress conditions during vascular disease. In this report, using fluorescently-labeled, specific antibodies against a mouse platelet and neutrophil marker, we describe a detailed intravital microscopic protocol to monitor heterotypic interactions of platelets and neutrophils on the activated endothelium during TNF-&alpha;-induced inflammation or following laser-induced injury in cremaster muscle microvessels of live mice.

Here we report an experimental technique of fluorescence intravital microscopy to visualize heterotypic platelet-neutrophil interactions on the activated endothelium during vascular inflammation and thrombus formation in live mice. This microscopic technology will be valuable to study the molecular mechanism of vascular disease and to test pharmacologic agents under pathophysiological conditions.

في الوقت الحقيقي التصوير من التفاعلات صفائح الدم، العدلات غيروي على البطانة الوعائية التهاب المنشط خلال تشكيل خثرة في والفئران الحية

Interaction of activated platelets and leukocytes (mainly neutrophils) on the activated endothelium mediates thrombosis and vascular inflammation.1,2 ...

A Flow Adhesion Assay to Study Leucocyte Recruitment to Human Hepatic Sinusoidal Endothelium Under Conditions of Shear Stress

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the development of fibrosis and progression to cirrhosis. Understanding the molecular mechanisms that mediate leucocyte recruitment to the liver could identify important therapeutic targets for liver disease. The key interaction during leucocyte recruitment is that of inflammatory cells with endothelium under conditions of shear stress. Recruitment to the liver occurs within the low shear channels of the hepatic sinusoids which are lined by hepatic sinusoidal endothelial cells (HSEC). The conditions within the hepatic sinusoids can be recapitulated by perfusing leucocytes through channels lined by human HSEC monolayers at specific flow rates. In these conditions leucocytes undergo a brief tethering step followed by activation and firm adhesion, followed by a crawling step and subsequent transmigration across the endothelial layer. Using phase contrast microscopy, each step of this &#39;adhesion cascade&#39; can be visualized and recorded followed by offline analysis. Endothelial cells or leucocytes can be pretreated with inhibitors to determine the role of specific molecules during this process.

Leucocyte recruitment to the liver occurs within the specialized channels of the hepatic sinusoids which are lined by unique hepatic sinusoidal endothelial cells. Phase contrast microscopy of leucocyte recruitment across human hepatic sinusoidal endothelium under conditions of physiological shear stress can facilitate the elucidation of the molecular mechanisms which underlie this process.

تدفق التصاق الفحص لدراسة توظيف الكرية البيضاء لالكبدي الإنسان جيبية البطانة Endothelium تحت ظروف إجهاد القص

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the ...

Human Neutrophil Flow Chamber Adhesion Assay

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion involves many different adhesion molecules including members of the &beta;2 integrin family and their counter-receptors of the ICAM family. Recently, naturally occurring genetic variants in both &beta;2 integrins and ICAMs are reported to be associated with autoimmune disease. Thus, the quantitative adhesive capacity of neutrophils from individuals with varying allelic forms of these adhesion molecules is important to study in relation to mechanisms underlying development of autoimmunity. Adhesion studies in flow chamber systems can create an environment with fluid shear stress similar to that observed in the blood vessel environment in vivo. Here, we present a method using a flow chamber assay system to study the quantitative adhesive properties of human peripheral blood neutrophils to human umbilical vein endothelial cell (HUVEC) and to purified ligand substrates. With this method, the neutrophil adhesive capacities from donors with different allelic variants in adhesion receptors can be assessed and compared. This method can also be modified to assess adhesion of other primary cell types or cell lines.

A method of quantitating neutrophil adhesion is reported. This method creates a dynamic flow environment similar to that encountered in a blood vessel. It allows the investigation of neutrophil adhesion to either purified adhesion molecules (ligand) or endothelial cell substrate (HUVEC) in a context similar to the in vivo environment with sheer stress.

غرفة تدفق العدلات الإنسان التصاق الفحص

Neutrophil firm adhesion to endothelial cells plays a critical role in inflammation in both health and disease. The process of neutrophil firm adhesion ...

Scalable High Throughput Selection From  Phage-displayed Synthetic Antibody Libraries

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. However, it is apparent that traditional monoclonal technologies are not alone up to this task. This has led to the development of alternate methods to satisfy the demand for high quality and renewable affinity reagents to all accessible elements of the proteome. Toward this end, high throughput methods for conducting selections from phage-displayed synthetic antibody libraries have been devised for applications involving diverse antigens and optimized for rapid throughput and success. Herein, a protocol is described in detail that illustrates with video demonstration the parallel selection of Fab-phage clones from high diversity libraries against hundreds of targets using either a manual 96 channel liquid handler or automated robotics system. Using this protocol, a single user can generate hundreds of antigens, select antibodies to them in parallel and validate antibody binding within 6-8 weeks. Highlighted are: i) a viable antigen format, ii) pre-selection antigen characterization, iii) critical steps that influence the selection of specific and high affinity clones, and iv) ways of monitoring selection effectiveness and early stage antibody clone characterization. With this approach, we have obtained synthetic antibody fragments (Fabs) to many target classes including single-pass membrane receptors, secreted protein hormones, and multi-domain intracellular proteins. These fragments are readily converted to full-length antibodies and have been validated to exhibit high affinity and specificity. Further, they have been demonstrated to be functional in a variety of standard immunoassays including Western blotting, ELISA, cellular immunofluorescence, immunoprecipitation and related assays. This methodology will accelerate antibody discovery and ultimately bring us closer to realizing the goal of generating renewable, high quality antibodies to the proteome.

A method is described with visual accompaniment for conducting scalable, high throughput selections from phage-displayed combinatorial synthetic antibody libraries against hundreds of antigens simultaneously. Using this parallel approach, we have isolated antibody fragments that exhibit high affinity and specificity for diverse antigens that are functional in standard immunoassays.

تحجيم إنتاجية عالية التحديد من المكتبات الأجسام المضادة الاصطناعية-عرض فاجي

The demand for antibodies that fulfill the needs of both basic and clinical research applications is high and will dramatically increase in the future. ...

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood.
To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

في المختبر والمجراة نموذج لدراسة التصاق البكتيريا إلى سفينة ستريت تحت ظروف التدفق

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

Intravital Microscopy of Leukocyte-endothelial and Platelet-leukocyte Interactions in Mesenterial Veins in Mice

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this protocol, we describe intravital microscopy of mesentery veins. This can be performed in a short period of time with reproducible results showing leukocyte-endothelial interactions in vivo. We describe an inflammatory setting after LPS challenge of the endothelium. But in this model one can apply many different types of inflammatory conditions, like bacterial, chemical or biological and investigate the administration of drugs and their direct effects on the living animal and its impact on leukocyte recruitment. This protocol has been applied successfully to a number of different treatments of mice and their effects on inflammatory response in vessels. Herein, we describe the visualization of leukocytes and platelets by fluorescently labeling these with rhodamine 6G. Additionally, any specific imaging can be performed using targeted fluorescently labeled molecules.

This simplified application of intravital microscopy of mesentery veins in mice can be used in different models of inflammation to observe leukocyte-endothelial and platelet-leukocyte interactions.

التفاعلات حيوي داخلي المجهري من خلايا الدم البيضاء-البطانية وصفائح الدم الكريات البيض في المساريقية الأوردة في الفئران

Intravital microscopy is a method that can be used to investigate different processes in different regions and vessels in living animals. In this ...

Assay of Adhesion Under Shear Stress for the Study of T Lymphocyte-Adhesion Molecule Interactions

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and interaction with antigen presenting cells (APC) in the formation of immunological synapses. These two contexts of T cell adhesion differ in that T cell-APC interactions can be considered static, while T cell-blood vessel interactions are challenged by the shear stress generated by circulation itself. T cell-APC interactions are classified as static in that the two cellular partners are static relative to each other. Usually, this interaction occurs within the lymph nodes. As a T cell interacts with the blood vessel wall, the cells arrest and must resist the generated shear stress.1,2 These differences highlight the need to better understand static adhesion and adhesion under flow conditions as two distinct regulatory processes. The regulation of T cell adhesion can be most succinctly described as controlling the affinity state of integrin molecules expressed on the cell surface, and thereby regulating the interaction of integrins with the adhesion molecule ligands expressed on the surface of the interacting cell. Our current understanding of the regulation of integrin affinity states comes from often simplistic in vitro model systems. The assay of adhesion using flow conditions described here allows for the visualization and accurate quantification of T cell-epithelial cell interactions in real time following a stimulus. An adhesion under flow assay can be applied to studies of adhesion signaling within T cells following treatment with inhibitory or stimulatory substances. Additionally, this assay can be expanded beyond T cell signaling to any adhesive leukocyte population and any integrin-adhesion molecule pair.

This flow adhesion assay provides a simple, high impact model of T cell-epithelial cell interactions. A syringe pump is used to generate shear stress, and confocal microscopy captures images for quantification. The goal of these studies is to effectively quantify T cell adhesion using flow conditions.

فحص من التصاق تحت إجهاد القص لدراسة تي اللمفاويات-التصاق جزيء التفاعلات

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and ...