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

Эта работа финансировалась NIH RO1 AI079253 и Департамент по делам ветеранов.

1. Животные модели <ol><li> Мыши: Используйте phox p47-/ - мышей и возрасту и полу C57BL6/DBA мышей. Получить разрешение на эксперименты с Институциональные уходу и использованию животных комитета. </li><li> Анестезия: Используйте непрерывную изофлуран системы управления для анестезии. Сист?...

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&quot;Реального времени&quot; измерения активных форм кислорода (АФК) в живых животных может быть достигнуто с помощью флуоресцентных зондов и хемилюминесцентных. В то время как флуоресцентные зонды страдают от того, слабая сигнал-шум 12, изображениями методике, описанной более чувств...

<table border="1"><tbody><tr><td> Название реагента </td><td> Компания </td><td> Номер в каталоге </td><td> Комментарии </td></tr><tr><td> L-012 </td><td> Wako Chemicals USA, Inc </td><td> 120-04891 </td><td></td></tr><tr><td> Зимозан </td><td> Sigma, Сент-Луис, Миссури </td><td> Z4250 </td><td></td></tr><tr><td> четыреххлористого углерода </td><td> Sigma, Сент-Луис, Миссури </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 оксидазы является основным источником активных форм кислорода (АФК) в фагоцитов. Из-за эфемерность ROS, это трудно для измерения и мониторинга уровня АФК в живых животных. Минимально инвазивных методов для последовательного количественного АФК в живых мышей описано.

Биолюминесценция Визуализация активности NADPH оксидазы на различных животных моделях

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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