Sterile inflammation (SI), inflammation in the absence of microbial pathogens, is responsible for the morbidity and mortality of millions of Americans every year. Decades of pathologic research have shown that innate immune cells play a critical role in the initiation and resolution of SI; however, researchers do not understand why SI exists or what stimulates this process. A barrier to progress in the field has been the lack of a suitable system with which to image SI intra-vitally. In vitro systems poorly recapitulate complex tissue-level inflammatory responses, and murine models require surgical procedures to perform intra-vital imaging. To address these issues, we have developed a system with which to study SI in zebrafish larvae.
Zebrafish larvae are an ideal system in which to study SI because their small size and transparency allows for intravital imaging without causing damage to the animal. We adapted the Nitroreductase/Metronidazole (NTR-Mtz) system in order to induce tissue damage via production of reactive oxygen species (ROSs) in the livers of zebrafish larvae. Transgenic leukocyte reporter lines and fluorescent dyes were crossed with animals that express NTR in hepatocytes and imaged intra-vitally using our spinning disk confocal microscope.
We found that the NTR/Mtz system induces apoptosis and necrosis in hepatocytes within 24hrs of Mtz exposure, and that reactive oxygen species (ROSs) are present during this process. Leukocyte recruitment to the liver corresponds with the onset of these cell death processes. Furthermore, neutrophils and macrophages show distinct behaviors in both the timing of their recruitment and their morphology upon arriving to the damaged liver. Neutrophils appear to patrol the liver, and may undergo cell death prior to being engulfed by nearby leukocytes and hepatocytes. Macrophages appear to patrol the liver and phagocytose (presumably damaged) hepatocytes. This system has enabled us to observe cell death and inflammation in vivo and in real time in the larval zebrafish liver. These processes may be inhibited by pharmacologic or genetic perturbation. We believe that this adaptation of the NTR/Mtz system will enable progress in the field of SI.
|Commitee:||Chu, Jaime, Evans, Todd, Overholtzer, Michael, Zallen, Jennifer|
|School:||Weill Medical College of Cornell University|
|Department:||Cell & Developmental Biology|
|School Location:||United States -- New York|
|Source:||DAI-B 80/07(E), Dissertation Abstracts International|
|Subjects:||Biology, Pathology, Physiology|
|Keywords:||Leukocytes, Liver, Liver injury, Nitroreductase metronidazole, Sterile inflammation|
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