Name: bioluminescence imaging of nadph oxidase activity in different animal models
Uploaded: 2012-10-22T12:00:00
Description: Watch this Scientific Journal Video about Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models at JoVE.com

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: اس...

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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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