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

इस काम NIH RO1 AI079253 और दिग्गजों मामलों के विभाग द्वारा वित्त पोषित किया गया था.

1. पशु मॉडल <ol><li> चूहे: p47 phox / प्रयोग चूहों और उम्र और सेक्स मिलान C57BL6/DBA चूहों. प्रयोगों के लिए संस्थागत पशु की देखभाल और उपयोग समिति से अनुमोदन प्राप्त करने के. </li><li> संज्ञाहरण: एक निरंतर isoflurane प्रशासन प्र...

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&quot;वास्तविक समय&quot; प्रतिक्रियाशील ऑक्सीजन प्रजातियों (ROS) के रहने वाले जानवरों में माप फ्लोरोसेंट और chemiluminescent जांच का उपयोग करके प्राप्त किया जा सकता है. जबकि फ्लोरोसेंट जांच कमजोर संकेत से शोर अनुपात 12</...

<table border="1"><tbody><tr><td> अभिकर्मक के नाम </td><td> कंपनी </td><td> कैटलॉग संख्या </td><td> टिप्पणियां </td></tr><tr><td> एल-012 </td><td> Wako रसायन यूएसए इंक, </td><td> 120-04891 </td><td></td></tr><tr><td> Zymosan </td><td> सिग्मा, सेंट लुइस, MO </td><td> Z4250 </td><td></td></tr><tr><td> कार्बन टेट्राक्लोराइड </td><td> सिग्मा, सेंट लुइस, 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 oxidase प्रतिक्रियाशील ऑक्सीजन प्रजातियों phagocytes (ROS) के प्रमुख स्रोत है. ROS के अल्पकालिक प्रकृति की वजह से, यह मुश्किल है करने के लिए उपाय करने के लिए और रहने वाले जानवरों में ROS के स्तर की निगरानी. रहने वाले चूहों में ROS के धारावाहिक मात्रा का ठहराव के लिए एक न्यूनतम इनवेसिव विधि का वर्णन है.

NADPH oxidase गतिविधि के विभिन्न पशु मॉडल में bioluminescence इमेजिंग

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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