05; n = 5 in each group

Statistical significance using the Mann-Whitney test (*P <0.05; n = 5 in each group).Takahashi et al. Critical Care 2013, 17:R160 http://ccforum.com/content/17/4/RPage 7 ofA/50 imagesAutophagosomeAutolysosome *P= 0.NS0 Sham CLP Sham CLPBFigure 3 Electron microscopic analysis of the liver. (A) The number of autophagosomes and autolysosomes are compared in CLP and sham animals. All data are expressed as the mean ?SD. Data were analyzed for statistical significance using the Mann-Whitney test. Increase in autolysosomes in the CLP group was statistically significant (*P <0.05; n = 3); mean increases in autophagosomes in CLP compared to sham did not 2-Bromo-4-fluoro-5-methylbenzoic acid reach statistical significance. NS, not significant. (B) Images of electron microscopy of the liver; a: Liver sample obtained from sham-operated mice. Organelles in the hepatocyte are generally intact and lysosomes do not contain discrete PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/1956868 membrane structures, although the inhomogeneous electron-dense material often seen in (hetero)lyosomes most certainly represent end-stage degradation of phospholipid and other cytoplasmic materials (a material at the light microscopic level referred to as lipofuscin); b-e: CLP-operated mice. Double arrow heads identify complex structures bounded by two membranes (autophagosomes); arrow heads identify single membrane-bound lysosomal complexes with degraded organellar content (autolysosomes); e: the double arrow head identifies an autophagosome that clearly contains an injured mitochondrion.Takahashi et al. (E)-1,2-Di(pyridin-4-yl)ethene Critical Care 2013, 17:R160 http://ccforum.com/content/17/4/RPage 8 ofWe next evaluated liver injury by histology and serum transaminase levels. In sham-operated mice with chloroquine treatment, no liver damage was observed. In contrast, we observed mid-zonal sinusoidal congestion and dilatation at 6 h after CLP. The congestion and dilatation became greater in CLP mice given chloroquine treatment, and was associated with subsequent liver dysfunction (Figure 5A). Serum AST and ALT were modestly increased at 6 and 24 h after CLP, but was significantly elevated compared to sham and untreated CLP animals after treatment with chloroquine (Figure 5B). Finally, we examined the survival of CLP mice treated with or without chloroquine. Mice with labored breathing were considered moribund and were euthanized. Up to 36 h after CLP, the number of moribund mice in the chloroquine-treated group was significantly greater than that in the untreated group (Figure 5C; P = 0.003). From these data, it is evident that suppression of autophagy accelerates liver injury, and likely contributes to the increased mortality in the CLP septic model, thus suggesting that induction of autophagy plays a protective role against sepsis in this model.Discussion In this study, we investigated the kinetics and role of autophagy in septic C57BL/6N mice over a 24-h period following CLP. We augmented our analysis by taking advantage of the unique characteristics of CLP-treated PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/22993420 GFP-LC3 transgenic mice, in which LC3-positive autophagosomes can be directly visualized by GFP. Autophagosome formation as assessed by LC3-I/LC3-II conversion and GFP-LC3 dots was detected in liver, heart, and spleen, peaking at 6 h after CLP. These findings are corroborated by other recent reports of increased autophagy in the heart, liver, and lungs of both CLP-treated animals and 2-methyl-4-oxo- in patients with sepsis [20-22,28]. Importantly, the time sequence of autophagy in these studies, with peak autophagosome formation at 6 to 8 h af.