وقد تم تمويل هذا العمل من قبل المعاهد الوطنية للصحة RO1 AI079253 وزارة شؤون المحاربين القدامى.

1. نماذج الحيوان <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> الفئران: استخدام P47-phox / - الفئران والعمر والجنس المتطابقة-الفئران C57BL6/DBA. الحصول على موافقة لإجراء التجارب من رعاية الحيوان واللجنة المؤسسية الاستخدام. </li><li style=";text-align:right;direction:rtl"> التخدير: استخدام نظام إدارة isoflurane المستمر للحث على التخدير. يتم تعبئة النظام المرذاذ (VetEquip) مع isoflurane (2-3٪). تؤكد أن الفئران هي تخدير كامل من خلال مراقبة حركة التنفس، ومنعكس القرنية استجابة للمؤثرات الخارجية. </li><li style=";text-align:right;direction:rtl"> العمليات الجراحية: فرك منطقة العمليات الجراحية (الفوق) مع الايثانول 70٪. ارتداء قفازات معقمة ونظيفة ثوب الجراحية والقناع. إعداد الفئران عن طريق تقليم الشعر وتطبيق betadine بالتناوب مع الايثانول 70٪ إلى المنطقة حيث سيتم شق. إجراء عمليات جراحية على سطح معقمة واستخدام أدوات معقمة. رصد الفئران بعد العملية حتى مستيقظا ويتحرك بحرية. </li><li style=";text-align:right;direction:rtl"> داخل الرغامى زيموزان الإدارة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إدارة التخديركما هو موضح في 1.2. </li><li style=";text-align:right;direction:rtl"> تطهير منطقة العمليات الجراحية (الرقبة) مع betadine والايثانول 70٪ وفضح القصبة الهوائية عن طريق تشريح الجراحي. </li><li style=";text-align:right;direction:rtl"> بيرس القصبة الهوائية مع إبرة قياس 27-وضخ زيموزان (سانت لويس، MO) بجرعة من 1 ميكروغرام / غرام باستخدام ميكروغرام 0.5 / ميكرولتر حل (المجموع 50 ميكرولتر حجم للماوس ز 25) الذائب في PBS العقيمة. </li><li style=";text-align:right;direction:rtl"> إغلاق الجرح الفئران الرقبة مع خيوط الحرير 5-0 في ظل ظروف معقمة العقيم. </li><li style=";text-align:right;direction:rtl"> الصورة بعد 4 ساعات وعلى مدار 24 ساعة. </li></ol></li><li style=";text-align:right;direction:rtl"> الأعور ربط وثقب <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إدارة التخدير كما هو موضح في 1.2. </li><li style=";text-align:right;direction:rtl"> مقطع الشعر في منطقة العمليات الجراحية (البطن) وتعقيمها مع betadine والايثانول 70٪. </li><li style=";text-align:right;direction:rtl"> تنفيذ خط الوسط البطن وتحديد الأعور. </li><li style=";text-align:right;direction:rtl"> Ligate على القاصي 50٪ من المعرضين الأعور مع خياطة الحرير 4-0 و الأعور ثقب البعيدة للربط مع مرور واحد من إبرة قياس 21-. </li><li style=";text-align:right;direction:rtl"> إغلاق شق مع خيوط الحرير 4-0 العقيمة. </li><li style=";text-align:right;direction:rtl"> الصورة بعد 4 ساعات و24 ساعة. </li></ol></li><li style=";text-align:right;direction:rtl"> رابع كلوريد الكربون عن طريق الفم (لجنة علم المناخ 4) الإدارة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إدارة التخدير كما هو موضح في 1.2. </li><li style=";text-align:right;direction:rtl"> إدارة لجنة علم المناخ 4 (2 ميكروغرام / غرام، سانت لويس، MO) المذاب في زيت الذرة بالتزقيم عن طريق الفم. يجب أن يتم تنفيذ الإجراء من CCL4 الإدارة في مجال معين وفقا للIBC / EHS السياسات. </li><li style=";text-align:right;direction:rtl"> الصورة بعد 4 ساعات وعلى مدار 24 ساعة. </li></ol></li></ol> 2. الحصول على صورة Bioluminescent <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تهيئة ال 200 IVIS نظام التصوير (Xenogen شركة، ألاميدا، CA)، تعيين إلى وضع التلألؤ، وانتظر الجهاز المسؤول عن يقترن (CCD) لتأمين درجة حرارة. </li><li style=";text-align:right;direction:rtl"> حدد وقت التعرض، binning (وسط) وF / إيقاف إعدادات (8). </li><li style=";text-align:right;direction:rtl"> الفئران في موقف ضعيف مكان في غرفة التصوير على مسرح ساخنة للحفاظ على درجة حرارة الجسم طبيعية. إدارة التخدير كما هو موضح في 1.2 عن طريق الأنف بينما مخروط الفئران هي في غرفة التصوير IVIS. </li><li style=";text-align:right;direction:rtl"> إدارة L-012<html> (20 ميكروغرام / غرام) في حل PBS العقيمة (10 ميكروغرام / ميكرولتر) عن طريق الوريد عن طريق الحقن خلف الحجاج في كل نقطة الوقت المحدد للتصوير. </li><li style=";text-align:right;direction:rtl"> التقاط الصور في بداية 2 دقيقة بعد الحقن L-012. نستخدم عادة التعرض و 40 ثانية. </li></ol> 3. تحليل البيانات باستخدام منطقة الاهتمام <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تحليل البيانات باستخدام البرامج الحية صورة v.4.2 (Xenogen شركة، ألاميدا، CA). </li><li style=";text-align:right;direction:rtl"> فتح صورة وحدد قياس المنطقة من الأدوات الفائدة. </li><li style=";text-align:right;direction:rtl"> تحديد المنطقة من شكل وحجم الفائدة على / أو الصدر والبطن بعد تتراكب صورة ملونة شبه الرقمية (التي تمثل كشف الفوتون) على صورة فوتوغرافية من الفأرة. </li><li style=";text-align:right;direction:rtl"> تحديد كثافة إشارة (الفوتون تدفق) من منطقة الفائدة. </li></ol> 4. إحصائيات <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> استخدام حزمة برامج التحليل الإحصائي لأداء اتجاهين مع ANOVA Bonferroni بعد الاختبار في نقاط زمنية الفردية. </li></ol>TLE &quot;&gt; نتائج الممثل 5. زيموزان هي خلايا الخميرة الموالية للالتهابات المنتج الجدار ومنشط قوي للأوكسيديز NADPH 3. أظهرنا سابقا أن زيموزان داخل الرغامى يدفع أكثر قوة والتهاب الرئة عدلة الموالية للالتهابات إنتاج السيتوكينات في P47-phox / - مقارنة wildtype الفئران 1. هنا، كان هدفنا لمقارنة ROS الجيل في الرئتين من wildtype وP47-phox / - الفئران بعد التحدي زيموزان. في 4 ساعات بعد تناوله زيموزان داخل الرغامى، لاحظنا زيادة كبيرة في الانبعاثات الفوتون على الصدر في الفئران مقارنة مع wildtype الأساس، وكذلك زيادة في الانبعاثات الفوتون في wildtype مقارنة P47-phox / - الفئران في 4 ساعات وعلى مدار 24 ساعة. في المقابل، إشارات تلألؤ بيولوجي في P47-phox / - الفئران ظلت عند مستويات خط الأساس في 4 ساعات بعد العلاج زيموزان (Fig.1 AB). وهكذا، يبدو أن أوكسيديز NADPH به المصدر الرئيسي للجيل ROS في الرئتين بعد زيموزان الإدارة. المقبل، قمنا بتقييم ROS الإنتاج في نموذج الإنتان CLP-المستحث. الإنتان هو متلازمة تهدد الحياة المرتبطة نهاية الجهاز الضرر، بما في ذلك الضرر الرئة الحاد (ALI) ومتلازمة الضائقة التنفسية الحادة (ARDS) 4، 5. وقد اعتبر ROS بوساطة الإصابة ليكون عاملا هاما القيادة الإنتان الناجم عن اختلال وظيفي متعدد الجهاز. وجدنا أن زيادة كبيرة على مدى الصدر ROS (4 ساعة) وعلى البطن (24 ساعة) بعد CLP في الفئران مقارنة مع wildtype خط الأساس. غير أن مستويات ROS في P47-phox / - الفئران كانت مماثلة لمستويات خط الأساس في نقاط زمنية على حد سواء (Fig.2 AC). هذه النتائج تبين أن الجيل ROS في نموذج الفأر الإنتان الناجم عن CLP-NADPH أوكسيديز هو المعتمدة. وأخيرا، كنا bioluminescent التصوير للكشف عن ROS الإنتاج في 4-الناجم عن الإصابة جنة علم المناخ الكبد الوضعلتر. 4 أسباب نخر جنة علم المناخ الخلايا الكبدية، واستخدمت في تصميم نموذج كل من اصابة الكبد الحاد وتليف الكبد 6. وعلى النقيض من النماذج التي سبق وصفها من الالتهاب والإصابة، وجدنا أن ROS الإنتاج في البطن كان متواضعا (حوالي 35٪) أكثر من خط الأساس في زيادة P47-phox / - الفئران في 4 ساعات بعد 4 للجنة علم المناخ الإدارة. ومع ذلك، كان حجم لجنة علم المناخ الناجم 4-ROS جيل أكبر بكثير على الفئران في P47 wildtype من phox / - الفئران (Fig.3 AB). هذه البيانات تشير إلى أن كلا الجيل ROS P47 phox التي تحتوي على NADPH أوكسيديز التي تعتمد على ومستقلة تحدث في لجنة علم المناخ الحاد الناجم عن إصابة 4-الكبد. <img alt="الشكل 1" src="/files/ftp_upload/3925/3925fig1.jpg" /> الرقم الإنتاج ROS 1. في الرئتين بعد العلاج زيموزان. A) الممثل bioluminescenم التصوير الإنتاج ROS. ملاحظة أنه بالإضافة إلى صدره، والتلألؤ قابلا للاكتشاف على البطن في كل من wildtype وP47-phox / - الفئران والتلألؤ البطن هو دون تغيير عن طريق العلاج زيموزان. B) من التهم الفوتون صدر wildtype وP47-phox / - الفئران بعد حقنة واحدة داخل الرغامى (IT) من زيموزان. تم إجراء التصوير تلألؤ بيولوجي في الأساس، ح 4، و24 ساعة. وقد تم تحديد انبعاث الضوء من منطقة الفوائد على الصدر باستخدام المشار شبه لون الحجم. يتم عرض النتائج على النحو يعني ± SE، ن = 6-9 في المجموعة، * = P &lt;0.05. <a href="http://www.jove.com/files/ftp_upload/3925/3925fig1large.jpg" target="_blank">اضغط هنا لتكبير الرقم</a> . <img alt="الشكل 2" src="/files/ftp_upload/3925/3925fig2.jpg" /> الكشف عن الرقم ROS 2. بعد CLP. A تلألؤ بيولوجي الممثل) التصوير الإنتاج ROS، B) من التهم الفوتون الصدر، وC) التهم الفوتون من بطن wildtype وP47-phox / - الفئران في الأساس، 4 ساعة، وبعد 24 ساعة CLP. يتم عرض النتائج على النحو يعني ± SE، ن = 4-5 في المجموعة، * = P &lt;0.05. <a href="http://www.jove.com/files/ftp_upload/3925/3925fig2large.jpg" target="_blank">اضغط هنا لتكبير الرقم</a> . <img alt="الشكل 3" src="/files/ftp_upload/3925/3925fig3.jpg" /> إنتاج الرقم ROS 3. في البطن بعد لجنة علم المناخ الناجم عن إصابة 4-الكبد. A) التصوير تلألؤ بيولوجي الممثل الإنتاج ROS وB) التهم الفوتون من بطن wildtype وP47-phox / - الفئران في الأساس، والموارد البشرية 4، وعلى مدار 24 ساعة عن طريق الفم بعد العلاج 4 للجنة علم المناخ. يتم عرض النتائج على النحو يعني ± SE؛ ن = 4-5 في المجموعة، * = P &lt;0.05 <a href="http://www.jove.com/files/ftp_upload/3925/3925fig3large.jpg" target="_blank">انقر هنا للحصول على أكبر شخصية</a> </A&gt;.

<ol>
	<li>Segal, B., Han, W., Bushey, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NADPH+oxidase+limits+innate+immune+response+in+the+lungs+in+mice.">NADPH oxidase limits innate immune response in the lungs in mice.</a> PLoS ONE. 5, e9631 (2010).</li><li>Jackson, S., Gallin, J., Holland, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+p47phox+mouse+knock-out+model+of+chronic+granulomatous+disease.">The p47phox mouse knock-out model of chronic granulomatous disease.</a> J. Exp. Med. 182, 751-758 (1995).</li><li>Gantner, B. N., Simmons, R. M., Underhill, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collaborative+induction+of+inflammatory+responses+by+dectin-1+and+Toll-like+receptor+2.">Collaborative induction of inflammatory responses by dectin-1 and Toll-like receptor 2.</a> J. Exp. Med. 197, 1107-1117 (2003).</li><li>Dejager, L., Pinheiro, I., Libert, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cecal+ligation+and+puncture:+the+gold+standard+model+for+polymicrobial+sepsis.">Cecal ligation and puncture: the gold standard model for polymicrobial sepsis.</a> Trends Microbiol. 19, 198-208 (2011).</li><li>Tsiotou, A. G., Sakorafas, G. H., Bramis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Septic+shock;+current+pathogenetic+concepts+from+a+clinical+perspective.">Septic shock; current pathogenetic concepts from a clinical perspective.</a> Med. Sci. Monit. 11, 76-85 (2005).</li><li>Fujii, T., Fuchs, B. C., Tanabe, 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=Mouse+model+of+carbon+tetrachloride+induced+liver+fibrosis:+Histopathological+changes+and+expression+of+CD133+and+epidermal+growth+factor.">Mouse model of carbon tetrachloride induced liver fibrosis: Histopathological changes and expression of CD133 and epidermal growth factor.</a> BMC Gastroenterology. 10, 79-90 (2010).</li><li>Kubo, H., Morgensterm, D., Doerschuk, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preservation+of+complement-induced+lung+injury+in+mice+with+deficiency+of+NADPH+oxidase.">Preservation of complement-induced lung injury in mice with deficiency of NADPH oxidase.</a> J. Clin. Invest. 97, 2680-2684 (1996).</li><li>Segal, B., Sakamoto, N., Bulkley, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xanthine+oxidase+contributes+to+host+defense+against+Burkholderia+cepacia+in+the+p47(phox-/-)+mouse+model+of+chronic+granulomatous+disease.">Xanthine oxidase contributes to host defense against Burkholderia cepacia in the p47(phox-/-) mouse model of chronic granulomatous disease.</a> Infect Immun. 68, 2374-2378 (2000).</li><li>Droge, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Free+radicals+in+the+physiological+control+of+cell+function.">Free radicals in the physiological control of cell function.</a> Physiological reviews. 82, 47-95 (2002).</li><li>Jones, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radical-free+biology+of+oxidative+stress.+American+journal+of+physiology.">Radical-free biology of oxidative stress. American journal of physiology.</a> Cell physiology. 295, C849-C868 (2008).</li><li>Hubbard, W. J., Choudhry, M., Chaudry, I. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cecal+ligation+and+puncture.">Cecal ligation and puncture.</a> Shock. 24, 52-57 .</li><li>Wardman, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+and+luminescent+probes+for+measurement+of+oxidative+and+nitrosative+species+in+cells+and+tissues:+progress,+pitfalls,+and+prospects.">Fluorescent and luminescent probes for measurement of oxidative and nitrosative species in cells and tissues: progress, pitfalls, and prospects.</a> Free Radic. Biol. Med. 43, 995-1022 (2007).</li><li>Pollock, J. D., Williams, D. A., Gifford, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+model+of+X-linked+chronic+granulomatous+disease,+an+inherited+defect+in+phagocyte+superoxide+production.">Mouse model of X-linked chronic granulomatous disease, an inherited defect in phagocyte superoxide production.</a> Nat. Genet. 9, 202-209 (1995).</li><li>Aramaki, Y., Y, ., Yoshida, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+sensitive+chemiluminescence+probe,+L-012,+for+measuring+the+production+of+superoxide+anion+by+cells.">A new sensitive chemiluminescence probe, L-012, for measuring the production of superoxide anion by cells.</a> Biochem. Biophys. Res. Commun. 193, 554-559 (1993).</li><li>Daiber, A., Oelze, M., August, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+superoxide+and+peroxynitrite+in+model+systems+and+mitochondria+by+the+luminol+analogue+L-012.">Detection of superoxide and peroxynitrite in model systems and mitochondria by the luminol analogue L-012.</a> Free Radic. Res. 38, 259-269 (2004).</li><li>Kielland, A., Blom, T., Nandakumar, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+reactive+oxygen+and+nitrogen+species+in+inflammation+using+the+luminescent+probe+L-012.">In vivo imaging of reactive oxygen and nitrogen species in inflammation using the luminescent probe L-012.</a> Free Radic. Biol. Med. 47, 760-766 (2009).</li></ol>

الإعلان عن أي تضارب في المصالح.

لا يمكن أن يتحقق &quot;في الوقت الحقيقي&quot; قياس أنواع الاكسجين التفاعلية (ROS) في الحيوانات الحية باستخدام تحقيقات الفلورسنت وchemiluminescent. في حين تحقيقات الفلورسنت يعانون من ضعف وجود إشارة إلى الضوضاء نسب 12، تقنية التصوير صفها هو أكثر حساسية للكشف عن انبعاث الضوء بعد...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم كاشف </td><td> شركة </td><td> كتالوج رقم </td><td> تعليقات </td></tr><tr><td> L-012 </td><td> واكو كيماويات الولايات المتحدة الأمريكية، وشركة </td><td> 120-04891 </td><td></td></tr><tr><td> زيموزان </td><td> سيغما، وسانت لويس، MO </td><td> Z4250 </td><td></td></tr><tr><td> رابع كلوريد الكربون </td><td> سيغما، وسانت لويس، MO </td><td> 289116 </td><td></td></tr></tbody></table>

bioluminescence imaging of nadph oxidase activity in different animal models

NADPH oxidase is a critical enzyme that mediates antibacterial and antifungal host defense. In addition to its role in antimicrobial host defense, NADPH oxidase has critical signaling functions that modulate the inflammatory response 1. Thus, the development of a method to measure in "real-time" the kinetics of NADPH oxidase-derived ROS generation is expected to be a valuable research tool to understand mechanisms relevant to host defense, inflammation, and injury.
Chronic granulomatous disease (CGD) is an inherited disorder of the NADPH oxidase characterized by severe infections and excessive inflammation. Activation of the phagocyte NADPH oxidase requires translocation of its cytosolic subunits (p47phox, p67phox, and p40phox) and Rac to a membrane-bound flavocytochrome (composed of a gp91phox and p22phox heterodimer). Loss of function mutations in any of these NADPH oxidase components result in CGD. Similar to patients with CGD, gp91phox -deficient mice and p47phox-deficient mice have defective phagocyte NADPH oxidase activity and impaired host defense 2, 13. In addition to phagocytes, which contain the NADPH oxidase components described above, a variety of other cell types express different isoforms of NADPH oxidase.
Here, we describe a method to quantify ROS production in living mice and to delineate the contribution of NADPH oxidase to ROS generation in models of inflammation and injury. This method is based on ROS reacting with L-012 (an analogue of luminol) to emit luminescence that is recorded by a charge-coupled device (CCD). In the original description of the L-012 probe, L-012-dependent chemiluminescence was completely abolished by superoxide dismutase, indicating that the main ROS detected in this reaction was superoxide anion 14. Subsequent studies have shown that L-012 can detect other free radicals, including reactive nitrogen species 15, 16. Kielland et al. 16 showed that topical application of phorbol myristate acetate, a potent activator of NADPH oxidase, led to NADPH oxidase-dependent ROS generation that could be detected in mice using the luminescent probe L-012. In this model, they showed that L-012-dependent luminescence was abolished in p47phox-deficient mice.
We compared ROS generation in wildtype mice and NADPH oxidase-deficient p47phox-/- mice 2 in the following three models: 1) intratracheal administration of zymosan, a pro-inflammatory fungal cell wall-derived product that can activate NADPH oxidase; 2) cecal ligation and puncture (CLP), a model of intra-abdominal sepsis with secondary acute lung inflammation and injury; and 3) oral carbon tetrachloride (CCl4), a model of ROS-dependent hepatic injury. These models were specifically selected to evaluate NADPH oxidase-dependent ROS generation in the context of non-infectious inflammation, polymicrobial sepsis, and toxin-induced organ injury, respectively. Comparing bioluminescence in wildtype mice to p47phox-/- mice enables us to delineate the specific contribution of ROS generated by p47phox-containing NADPH oxidase to the bioluminescent signal in these models.
Bioluminescence imaging results that demonstrated increased ROS levels in wildtype mice compared to p47phox-/- mice indicated that NADPH oxidase is the major source of ROS generation in response to inflammatory stimuli. This method provides a minimally invasive approach for "real-time" monitoring of ROS generation during inflammation in vivo.

Watch this Scientific Journal Video about Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models at JoVE.com

Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models

NADPH أوكسيديز هو المصدر الرئيسي للأنواع الاكسجين التفاعلية (ROS) في البالعات. نظرا لطبيعة زائلة من ROS، فإنه من الصعب قياس ومراقبة مستويات ROS في الحيوانات الحية. يوصف أسلوب القياس الكمي للمينيملي المسلسل من ROS في الفئران الحية.

التصوير تلألؤ بيولوجي من نشاط إنزيم NADPH في مختلف النماذج الحيوانية

NADPH oxidase is a critical enzyme that mediates antibacterial and antifungal host defense. In addition to its role in antimicrobial host defense, NADPH ...

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JoVE Journal

Immunology and Infection

16.1K Views.  Vanderbilt University School of Medicine. NADPH أوكسيديز هو المصدر الرئيسي للأنواع الاكسجين التفاعلية (ROS) في البالعات. نظرا لطبيعة زائلة من ROS، فإنه من الصعب قياس ومراقبة مستويات ROS في الحيوانات الحية. يوصف أسلوب القياس الكمي للمينيملي المسلسل من ROS في الفئران الحية.

Video: Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models

Diagnosis of Ecto- and Endoparasites in Laboratory Rats and Mice

Internal and external parasites remain a significant concern in laboratory rodent facilities, and many research facilities harbor some parasitized animals. Before embarking on an examination of animals for parasites, two things should be considered. One: what use will be made of the information collected, and two: which test is the most appropriate. Knowing that animals are parasitized may be something that the facility accepts, but there is often a need to treat animals and then to determine the efficacy of treatment. Parasites may be detected in animals through various techniques, including samples taken from live or euthanized animals. Historically, the tests with the greatest diagnostic sensitivity required euthanasia of the animal, although PCR has allowed high-sensitivity testing for several types of parasite. This article demonstrates procedures for the detection of endo- and ectoparasites in mice and rats. The same procedures are applicable to other rodents, although the species of parasites found will differ.

This article describes various procedures for screening rats and mice to detect endo- or ectoparasitism. Several diagnostic assays will be demonstrated, both those suitable for use on live animals and those used after euthanasia of the animal. Photographs to aid in identification of rat and mouse parasites will be included.

تشخيص وEndoparasites Ecto في الجرذان والفئران المعملية

Internal and external parasites remain a significant concern in laboratory rodent facilities, and many research facilities harbor some parasitized ...

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

اللعاب، الغدد اللعابية، وجمع اللمف الدموي من القراد اللبود الكتفي

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, neutrophils, T cell subsets, B cells, natural killer (NK) and NKT cells. Intravital microscopy of the liver for monitoring immune cell migration is particularly challenging due to the high requirements regarding sample preparation and fixation, optical resolution and long-term animal survival. Yet, the dynamics of inflammatory processes as well as cellular interaction studies could provide critical information to better understand the initiation, progression and regression of inflammatory liver disease. Therefore, a highly sensitive and reliable method was established to study migration and cell-cell-interactions of different immune cells in mouse liver over long periods (about 6 hr) by intravital two-photon laser scanning microscopy (TPLSM) in combination with intensive care monitoring.
The method provided includes a gentle preparation and stable fixation of the liver with minimal perturbation of the organ; long term intravital imaging using multicolor multiphoton microscopy with virtually no photobleaching or phototoxic effects over a time period of up to 6 hr, allowing tracking of specific leukocyte subsets; and stable imaging conditions due to extensive monitoring of mouse vital parameters and stabilization of circulation, temperature and gas exchange.
To investigate lymphocyte migration upon liver inflammation CXCR6.gfp knock-in mice were subjected to intravital liver imaging under baseline conditions and after acute and chronic liver damage induced by intraperitoneal injection(s) of carbon tetrachloride (CCl4).
CXCR6 is a chemokine receptor expressed on lymphocytes, mainly on Natural Killer T (NKT)-, Natural Killer (NK)- and subsets of T lymphocytes such as CD4 T cells but also mucosal associated invariant (MAIT) T cells1. Following the migratory pattern and positioning of CXCR6.gfp+ immune cells allowed a detailed insight into their altered behavior upon liver injury and therefore their potential involvement in disease progression.

Stable intravital high-resolution imaging of immune cells in the liver is challenging. Here we provide a highly sensitive and reliable method to study migration and cell-cell-interactions of immune cells in mouse liver over long periods (about 6 hours) by intravital multiphoton laser scanning microscopy in combination with intensive care monitoring.

المدى الطويل حيوي داخلي Multiphoton المجهر التصوير من الخلايا المناعية في السليمة والمريضة الكبد عن طريق الفئران CXCR6.Gfp مراسل

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, ...

Isolation and Transplantation of Different Aged Murine Thymic Grafts.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and selection of T cells specific for self and foreign antigens. The approach entails implantation of thymic grafts derived from various aged mice into immunodeficient scid recipients. Over a relatively short period of time the recipients are fully reconstituted with T cells derived from the implanted thymus graft. Only thymocytes seeding the thymus at the time of isolation undergo selection and develop into mature T cells. As such, changes in the nature and specificity of the engrafted T cells as a function of age-dependent thymic events can be assessed. Although technical expertise is required for successful thymic transplantation, this method provides a unique strategy to study in vivo a wide range of pathologies that are due to or a result of aberrant thymic function and/or homeostasis.

This report provides a detailed description of transplanting murine thymi from different aged donor mice under the kidney capsule of immunodeficient mouse recipients. The goal of this approach is to model T cell development and thymic selection events in vivo.

العزل وزرع مختلفة المسنين الفئران التوتة الطعوم.

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and ...

Bioluminescence Imaging to Detect Late Stage Infection of African Trypanosomiasis

Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades the central nervous system (CNS). In vivo study of the late stage has been limited as traditional methodologies require the removal of the brain to determine the presence of the parasites.
Bioluminescence imaging is a non-invasive, highly sensitive form of optical imaging that enables the visualization of a luciferase-transfected pathogen in real-time. By using a transfected trypanosome strain that has the ability to produce late stage disease in mice we are able to study the kinetics of a CNS infection in a single animal throughout the course of infection, as well as observe the movement and dissemination of a systemic infection.
Here we describe a robust protocol to study CNS infections using a bioluminescence model of African trypanosomiasis, providing real time non-invasive observations which can be further analyzed with optional downstream approaches.

This manuscript describes the use of a bioluminescent strain of African trypanosomes to enable the tracking of late stage infection and demonstrates how in vivo live imaging can be used to visualize infections within the central nervous system in real-time.

تلألؤ بيولوجي التصوير لكشف العدوى المرحلة المتأخرة من داء المثقبيات الأفريقي

Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and reproducibility of this assay can be considered unstable, either because of user&#39;s errors or because of the sensitivity of NK cells to experimental manipulation. To eliminate these issues, a workflow that reduces them to a minimum was established and is presented here. To illustrate, we obtained blood samples, at various time points, from runners (n = 4) that were submitted to an intense bout of exercise. First, NK cells are simultaneously identified and isolated through CD56 tagging and magnetic-based sorting, directly from whole blood and from as little as one milliliter. The sorted NK cells are removed of any reagent or capping antibodies. They can be counted in order to establish an accurate NK cell count per milliliter of blood. Secondly, the sorted NK cells (effectors cells or E) can be mixed with 3,3&#39;-Diotadecyloxacarbocyanine Perchlorate (DiO) tagged K562 cells (target cells or T) at an assay-optimal 1:5 T:E ratio, and analyzed using an imaging flow-cytometer that allows for the visualization of each event and the elimination of any false positive or false negatives (such as doublets or effector cells). This workflow can be completed in about 4 h, and allows for very stable results even when working with human samples. When available, research teams can test several experimental interventions in human subjects, and compare measurements across several time points without compromising the data&#39;s integrity.

This work describes an advanced workflow for the accurate and fast determination of NK (Natural Killer) cell count and NK cell cytotoxicity in human blood samples.

تدفق تحليل Cytometric من القاتل الطبيعي خلية التحللي آخر في الدم الجامع البشري

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging

Burn infections continue to be an important cause of morbidity and mortality. The increasing emergence of multidrug-resistant (MDR) bacteria has led to the frequent failure of traditional antibiotic treatments. Alternative therapeutics are urgently needed to tackle MDR bacteria.
An innovative non-antibiotic approach, antimicrobial blue light (aBL), has shown promising effectiveness against MDR infections. The mechanism of action of aBL is not yet well understood. It is commonly hypothesized that naturally occurring endogenous photosensitizing chromophores in bacteria (e.g., iron-free porphyrins, flavins, etc.) are excited by aBL, which in turn produces cytotoxic reactive oxygen species (ROS) through a photochemical process.
Unlike another light-based antimicrobial approach, antimicrobial photodynamic therapy (aPDT), aBL therapy does not require the involvement of an exogenous photosensitizer. All it needs to take effect is the irradiation of blue light; therefore, it is simple and inexpensive. The aBL receptors are the endogenous cellular photosensitizers in bacteria, rather than the DNA. Thus, aBL is believed to be much less genotoxic to host cells than ultraviolet-C (UVC) irradiation, which directly causes DNA damage in host cells.
In this paper, we present a protocol to assess the effectiveness of aBL therapy for MDR Acinetobacter baumannii infections in a mouse model of burn injury. By using an engineered bioluminescent strain, we were able to noninvasively monitor the extent of infection in real time in living animals. This technique is also an effective tool for monitoring the spatial distribution of infections in animals.

In Vivo Investigation of Antimicrobial Blue Light Therapy for Multidrug-resistant Acinetobacter baumannii Burn Infections Using Bioluminescence Imaging

Infections caused by multidrug-resistant (MDR) bacterial strains have emerged as a serious threat to public health, necessitating the development of alternative therapeutics. We present a protocol to evaluate the effectiveness of antimicrobial blue light (aBL) therapy for MDR Acinetobacter baumannii infections in mouse burns by using bioluminescence imaging.

في فيفو التحقيق من مضادات الميكروبات العلاج الضوء الأزرق للمقاومة للأدوية المتعددة baumannii الراكدة حرق العدوى عن طريق تلألؤ بيولوجي التصوير

Burn infections continue to be an important cause of morbidity and mortality. The increasing emergence of multidrug-resistant (MDR) bacteria has led to ...

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo fluorescence optical imaging (OI), in two different mouse models of inflammation: a rheumatoid arthritis (RA) and a contact hypersensitivity reaction (CHR) model. Light with a wavelength in the near infrared (NIR) window (650 - 950 nm) allows a deeper tissue penetration and minimal signal absorption compared to wavelengths below 650 nm. The major advantages using fluorescence OI is that it is cheap, fast and easy to implement in different animal models.
Activatable fluorescent probes are optically silent in their inactivated states, but become highly fluorescent when activated by a protease. Activated MMPs lead to tissue destruction and play an important role for disease progression in delayed-type hypersensitivity reactions (DTHRs) such as RA and CHR. Furthermore, MMPs are the key proteases for cartilage and bone degradation and are induced by macrophages, fibroblasts and chondrocytes in response to pro-inflammatory cytokines. Here we use a probe that is activated by the key MMPs like MMP-2, -3, -9 and -13 and describe an imaging protocol for near infrared fluorescence OI of MMP activity in RA and control mice 6 days after disease induction as well as in mice with acute (1x challenge) and chronic (5x challenge) CHR on the right ear compared to healthy ears.

Non-invasive In Vivo Fluorescence Optical Imaging of Inflammatory MMP Activity Using an Activatable Fluorescent Imaging Agent

This paper explains the application of fluorescent imaging using an activatable optical imaging probe to visualize the in vivo activity of key matrix metalloproteinases in two different experimental models of inflammation.

غير الغازية<em&gt; في فيفو</em&gt; الإسفار التصوير البصري من مب النشاط التهابات باستخدام عامل التصوير الفلورسنت أكتيفاتابل

This paper describes a non-invasive method for imaging matrix metalloproteinases (MMP)-activity by an activatable fluorescent probe, via in vivo ...

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research investigating immunological responses against A. fumigatus has been limited by the lack of consistent and reliable assays for measuring the antifungal activity of specific immune cells in vitro. A new method is described to assess the antifungal activity of primary monocytes and neutrophils from human donors against A. fumigatus using FLuorescent Aspergillus REporter (FLARE) conidia. These conidia contain a genetically encoded dsRed reporter, which is constitutively expressed by live FLARE conidia, and are externally labeled with Alexa Fluor 633, which is resistant to degradation within the phagolysosome, thus allowing a distinction between live and dead A. fumigatus conidia. Video microscopy and flow cytometry are subsequently used to visualize the interaction between conidia and innate immune cells, assessing fungicidal activity whilst also providing a wealth of information on phagocyte migration, phagocytosis and the inhibition of fungal growth. This novel technique has already provided exciting new insights into the host-pathogen interaction of primary immune cells against A. fumigatus. It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.
It is important to note the laboratory setup required to perform this assay, including the necessary microscopy and flow cytometry facilities, and the capacity to work with human donor blood and genetically manipulated fungi. However, this assay is capable of generating large amounts of data and can reveal detailed insights into the antifungal response. This protocol has successfully been used to study the host-pathogen interaction of primary immune cells against A. fumigatus.

Live Imaging of Antifungal Activity by Human Primary Neutrophils and Monocytes in Response to A. fumigatus

Here, we describe a protocol to assess antifungal activity of primary human immune cells in real-time using fluorescent Aspergillus reporter conidia in conjunction with live-cell video microscopy and flow cytometry. Generated data provide insight into host cell-Aspergillus interactions such as fungicidal activity, phagocytosis, cell migration and inhibition of fungal growth.

التصوير المباشر لآخر مضاد من قبل العدلات الإنسان الأساسية وحيدات في الاستجابة لل<em&gt; A. فوميجاتوس</em

Aspergillus fumigatus is an opportunistic fungal pathogen causing invasive infections in immunocompromised hosts with a high case-fatality rate. Research ...

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome (HUS), colonize to the intestinal tract of humans. To study the detailed mechanism of EHEC colonization in vivo, it is essential to have animal models to monitor and quantify EHEC colonization. We demonstrate here a mouse-EHEC colonization model by transforming the bioluminescent expressing plasmid to EHEC to monitor and quantify EHEC colonization in living hosts. Animals inoculated with bioluminescence-labeled EHEC show intense bioluminescent signals in mice by detection with a non-invasive in vivo imaging system. After 1 and 2 days post infection, bioluminescent signals could still be detected in infected animals, which suggests that EHEC colonize in hosts for at least 2 days. We also demonstrate that these bioluminescent EHEC locate to mouse intestine, specifically in the cecum and colon, from ex vivo images. This mouse-EHEC colonization model may serve as a tool to advance the current knowledge of the EHEC colonization mechanism.

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

A detailed protocol of a mouse model for enterohemorrhagic E. coli (EHEC) colonization by using bioluminescence-labeled bacteria is presented. The detection of these bioluminescent bacteria by a non-invasive in vivo imaging system in live animals can advance our current understanding of EHEC colonization.

الكشف عن استعمار الإشريكيّة القولونية Enterohemorrhagic موريني واستضافة بنظام الإضاءة الحيوية غير الغازية في فيفو

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome ...