Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models

Summary

NADPH oxidase is the major source of reactive oxygen species (ROS) in phagocytes. Because of the ephemeral nature of ROS, it is difficult to measure and monitor ROS levels in living animals. A minimally invasive method for serial quantification of ROS in living mice is described.

Abstract

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 (p47^phox, p67^phox, and p40^phox) and Rac to a membrane-bound flavocytochrome (composed of a gp91^phox and p22^phox heterodimer). Loss of function mutations in any of these NADPH oxidase components result in CGD. Similar to patients with CGD, gp91^phox -deficient mice and p47^phox-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 p47^phox-deficient mice.

We compared ROS generation in wildtype mice and NADPH oxidase-deficient p47^phox-/- 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 p47^phox-/- mice enables us to delineate the specific contribution of ROS generated by p47^phox-containing NADPH oxidase to the bioluminescent signal in these models.

Bioluminescence imaging results that demonstrated increased ROS levels in wildtype mice compared to p47^phox-/- 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.

Protocol

1. Animal Models

1. Mice: Use p47^phox-/- mice and age- and sex-matched C57BL6/DBA mice. Obtain approval for experiments from Institutional Animal Care and Use Committee.
2. Anesthesia: Use a continuous isoflurane administration system to induce anesthesia. The vaporizer system (VetEquip) is filled with isoflurane (2-3%). Confirm that mice are fully anesthetized by observing respiration, movement, and corneal reflex in response to external stimuli.
3. Surgical procedures: Scrub the surgical area (benchtop) with 70% ethanol. Wear sterile gloves and a clean surgical gown and mask. Prepare the mice by clipping the hair and applyi....

Discussion

"Real-time" measurement of reactive oxygen species (ROS) in living animals can be achieved by using fluorescent and chemiluminescent probes. While fluorescent probes suffer from having weak signal-to-noise ratios ¹², the imaging technique described is more sensitive for detection of light emission following a chemical reaction of ROS with the luminol-based substrate L-012. Like all bioluminescent imaging techniques, this methodology is limited by wavelength-dependent light absorption and scatter by organs and .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
L-012	Wako Chemicals USA, Inc.	120-04891
Zymosan	Sigma, St. Louis, MO	Z4250
carbon tetrachloride	Sigma, St. Louis, MO	289116