• Ilias I Siempos, Hilaire C Lam, Yan Ding, Mary E Choi, Augustine M K Choi, and Stefan W Ryter.
• Department of Medicine, Division of Pulmonary and Critical Care Medicine, Brigham and Women's Hospital; First Department of Critical Care Medicine and Pulmonary Services, University of Athens Medical School, Evangelismos Hospital, Athens, Greece.
• J Vis Exp. 2014 Jan 1(84):e51066.

AbstractExperimental sepsis can be induced in mice using the cecal ligation and puncture (CLP) method, which causes polymicrobial sepsis. Here, a protocol is provided to induce sepsis of varying severity in mice using the CLP technique. Autophagy is a fundamental tissue response to stress and pathogen invasion. Two current protocols to assess autophagy in vivo in the context of experimental sepsis are also presented here. (I) Transgenic mice expressing green fluorescence protein (GFP)-LC3 fusion protein are subjected to CLP. Localized enhancement of GFP signal (puncta), as assayed either by immunohistochemical or confocal assays, can be used to detect enhanced autophagosome formation and, thus, altered activation of the autophagy pathway. (II) Enhanced autophagic vacuole (autophagosome) formation per unit tissue area (as a marker of autophagy stimulation) can be quantified using electron microscopy. The study of autophagic responses to sepsis is a critical component of understanding the mechanisms by which tissues respond to infection. Research findings in this area may ultimately contribute towards understanding the pathogenesis of sepsis, which represents a major problem in critical care medicine.

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