Impaired early nutrition influences the chance of developing metabolic disorders in later life. markers of OS (superoxide anion, antioxidant defenses), and SIPS (lipofuscin, p53, p21, p16, pRb/Rb, Acp53, sirtuin-1) were performed. CR in the OF group reduced microsteatosis, decreased levels of superoxide anion, and increased protein expression of catalase and superoxide dismutase. Moreover, CR decreased lipofuscin staining, p21, p53, Acp53, and p16 but increased pRb/Rb and sirtuin-1 protein expression. CR did not affect the NF group. These results suggest that CR reduces hepatic disorders induced by OF. diet (NF and OF groups) or to a CR diet induced by a reduction in daily food supply of 20% (based on the food intake of each group), which led to the establishment of the NFCR and OFCR groups. To perform all the experiments presented in this study, two consecutive series of reproductions were used, corresponding to 12 different litters. Only males from each group (NF (= 5C6), OF (= 5C6), NFCR (= 4C5), and OFCR (= 4C5)) were studied at seven months of age. After 6 h of fasting, mice were sacrificed, and the livers were then harvested and immediately frozen in nitrogen for western blot analyses or fixed in formol for histological analyses. 2.2. Evaluation of Hepatic Structure by Histological Analysis At seven months of life, the livers from the NF (= 5), OF (= 5), NFCR (= 5) and OFCR (= 5) groups were rapidly removed and fixed in formol as previously BLU9931 described . Equatorial cross-sections were paraffin-embedded and stained with hematoxylin and eosin (H/E) for hepatic structure evaluation and with Massons Trichrome to evaluate hepatic fibrosis. For all those histological analyses, the slides were observed blindly by the same experimenter (C.Y), and three images were captured for each animal. A pathologist (Prof. C. Sempoux) confirmed the histological observations. Hepatic fibrosis was quantified using ImageJ 1.50b (Java 1.8.0_60, National Institutes of Health, MD, USA) (http://rsbweb.nih.gov/ij), as previously described . 2.3. Detection of Superoxide Anion (O2??) by Chemiluminescence Liver O2?? production was evaluated at seven months of life in the NF (= 5), OF (= 5), NFCR (= 5), and OFCR (= 5) groups using oxidative fluorescent dye hydroethidine (2 M, Sigma-Aldrich) [22,23] as previously explained . Briefly, liver sections were stained with hydroethidine by incubating the sections in a light-protected humidified chamber at 37 C for 30 min. The sections were rinsed with phosphate-buffered saline (PBS) and mounted using Fluoromount-G mounting medium with 46-diamidino-2-phenylindole (DAPI; Interchim, France). The slides were observed blindly by the same experimenter (C.Y), and images were obtained using a laser scanning confocal microscope (Leica SP5) equipped with an argon laser. At least BLU9931 four hepatic sections were assessed per animal with a 514-nm long-pass filter and were evaluated with ImageJ software . 2.4. Detection of Oxidative DNA Double-Strand Break As previously explained , liver sections from 7-month-old mice from your NF (= 5), OF BLU9931 (= 5), NFCR (= 5) and OFCR (= 5) groups were stained with 53BP-1 (1/100, Abcam, #ab21083) overnight CSF2RA at 4 C. The sections were then washed with PBS and incubated for two hours with Alexa Fluor-488-conjugated donkey anti-rabbit IgG (1/200, Abcam, #ab150073). The sections were then rinsed with PBS and mounted using Fluoromount-G mounting medium with DAPI. A negative control was obtained by incubation only with secondary antibody. The slides were observed blindly using a fluorescence microscope (Nikon, Eclipse Ti2 Series) by the same experimenter (C.Y), and at least four hepatic BLU9931 sections were assessed per animal using ImageJ software . 2.5. Detection of Liver Senescence by Histological Staining A marker of SIPS , lipofuscin, was recognized by diastase periodic acid Schiff (d-PAS) resistance and Fontana Masson and Sudan Black B (SBB) staining of livers from NF (= 5), OF (= 5), NFCR (= 5), and OFCR (= 5) groups. For all those histological analyses, three images were captured for each animal. Lipofuscin staining was quantified using ImageJ software. A quantitative analysis was performed blindly by a single experimenter (C. Y), as previously explained . 2.6. Western Blotting Liver proteins from your NF (= 5), OF (= 5), NFCR (= 4) and OFCR (= 4C6) groups were extracted at.