The autolysosome, which has a single limiting membrane and contains cytoplasmic organellar mate rials at various stages of degradation, can be distin guished from the autophagosome by electron microscopy. The in crease in autolysosomes in hepatocytes from sham versus CLP mice per 50 images for most each mouse was statistically significant 6 h after CLP. These data indicated that the autophagy process is completed in sepsis, rather than blocked at the fusion step, consistent with the immu nofluorescence results. Importantly, despite an increased number of autophagosomes in septic samples, hepatocytes did not appear to be committed to cell death and the vast majority of mitochondria in both sham and CLP groups appeared normal.

Protective role of autophagy in the CLP septic model Since the autophagy machinery is activated after CLP, we examined whether this activation is beneficial or detrimental by inhibiting autophagy. Chloroquine, used Inhibitors,Modulators,Libraries primarily as an antimalarial drug, inhibits fusion of the autophagosome and lysosome by increasing autopha gosomal and lysosomal pH. We Inhibitors,Modulators,Libraries first confirmed that chloroquine suppressed au tophagy in our CLP model. With chloroquine treatment, the number of GFP LC3 dots and co localized GFP LC3 and LAMP1 were reduced after 24 h when compared to untreated animals in both CLP and sham operated co horts. Thus, chloroquine treatment suppressed the fusion of autophagosomes and lysosomes. We next evaluated liver injury by histology and serum transaminase levels. In sham operated mice with chloro quine treatment, no liver damage was observed.

In con trast, we observed mid zonal sinusoidal congestion and dilatation at 6 h after CLP. The congestion and dilata tion became greater in CLP mice given chloroquine treatment, and was associated with subsequent liver Inhibitors,Modulators,Libraries dysfunction. Serum AST and ALT were modestly increased at 6 and 24 h Inhibitors,Modulators,Libraries after CLP, but was sig nificantly elevated compared to sham and untreated CLP animals after treatment with chloroquine. Finally, we examined Inhibitors,Modulators,Libraries the survival of CLP mice treated with or without chloroquine. Mice with labored breath ing 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.

From these data, it is evident that suppression of autophagy accelerates liver Vandetanib solubility injury, and likely contributes to the in creased mortality in the CLP septic model, thus sug gesting 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 GFP LC3 transgenic mice, in which LC3 positive autopha gosomes can be directly visualized by GFP.