Mouse models of non-alcoholic fatty liver disease (NAFLD) are required to define therapeutic targets, but detailed time-resolved studies to establish a sequence of events are lacking. Here, we fed male C57Bl/6N mice a Western or standard diet over 48 weeks. Multiscale time-resolved characterization was performed using RNA-seq, histopathology, immunohistochemistry, intravital imaging, and blood chemistry; the results were compared to human disease. Acetaminophen toxicity and ammonia metabolism were additionally analyzed as functional readouts. We identified a sequence of eight key events: formation of lipid droplets; inflammatory foci; lipogranulomas; zonal reorganization; cell death and replacement proliferation; ductular reaction; fibrogenesis; and hepatocellular cancer. Functional changes included resistance to acetaminophen and altered nitrogen metabolism. The transcriptomic landscape was characterized by two large clusters of monotonously increasing or decreasing genes, and a smaller number of ‘rest-and-jump genes’ that initially remained unaltered but became differentially expressed only at week 12 or later. Approximately 30% of the genes altered in human NAFLD are also altered in the present mouse model and an increasing overlap with genes altered in human HCC occurred at weeks 30–48. In conclusion, the observed sequence of events recapitulates many features of human disease and offers a basis for the identification of therapeutic targets.

Hypoalbuminemia (HA) is frequently observed in systemic inflammatory diseases and in liver disease. However, the influence of HA on the pharmacokinetics and toxicity of compounds with high plasma albumin binding remained insufficiently studied. The ‘lack-of-delivery-concept’ postulates that HA leads to less carrier mediated uptake of albumin bound substances into hepatocytes and to less glomerular filtration; in contrast, the ‘concept-of-higher-free-fraction’ argues that increased concentrations of non-albumin bound compounds facilitate hepatocellular uptake and enhance glomerular filtration. To address this question, we performed intravital imaging on livers and kidneys of anesthetized mice to quantify the spatio-temporal tissue distribution of the mycotoxin ochratoxin A (OTA) based on its auto-fluorescence in albumin knockout and wild-type mice. HA strongly enhanced the uptake of OTA from the sinusoidal blood into hepatocytes, followed by faster secretion into bile canaliculi. These toxicokinetic changes were associated with increased hepatotoxicity in heterozygous albumin knockout mice for which serum albumin was reduced to a similar extent as in patients with severe hypoalbuminemia. HA also led to a shorter half-life of OTA in renal capillaries, increased glomerular filtration, and to enhanced uptake of OTA into tubular epithelial cells. In conclusion, the results favor the ‘concept-of-higher-free-fraction’ in HA; accordingly, HA causes an increased tissue uptake of compounds with high albumin binding and increased organ toxicity. It should be studied if this concept can be generalized to all compounds with high plasma albumin binding that are substrates of hepatocyte and renal tubular epithelial cell carriers.

This study was conducted to provide comprehensive information on the anatomical and histological features of the upper (UL) and lower (LL) lips of Rahmani sheep by gross examination, morphometric analysis in addition to Light and scanning electron microscope (SEM). Samples from normal healthy adult male sheep heads were collected directly after slaughtering. UL and LL were dissected, and specimens were collected for both light and SEM. The UL was longer approximately by one‐fold and thicker by one‐fold at the median and the oral angle areas, and by one‐ and half‐folds at the paramedian area than the LL. The free border of both lips was characterized rostrally by the presence of labial projections. By SEM the edges of the inner aspect of the lips and of the philtrum were distinguished by labial projections. These projections which surrounding the philtrum subdivided into polygonal areas with numerous keratinized scales especially at the apical parts which increased dorsally toward the nostril. Most of the openings of the upper labial salivary glands were volcanic crater‐shaped while that of the lower labial salivary glands were various shapes; round folded margin, rosette and whirlpool shaped. Histologically, the shape of the projection or papillae differs at the median and paramedian areas of the UL than the LL. However, there was no differences near the oral angle area. In conclusion, the shape, size and amount of keratinization of the papillae may offer efficiency to the lips during feeding process.

The aim of this study was to determine whether probiotic-feeding affected the expression of cathelicidins (CATHs), a major family of antimicrobial peptides, in response to lipopolysaccharides (LPS) challenge in the proventriculus and cecum of broiler chicks. One-day-old male Chunky broiler chicks were fed with or without 0.4% probiotics for 7 days (P-group and non-P-group, respectively). Then, they were orally challenged with no LPS (0-LPS), 1 μg LPS (1-LPS), or 100 μg LPS (100-LPS) (n＝5 in all groups) in Experiment 1, and with no LPS or 1 μg LPS (n＝6 in all groups) in Experiment 2. Five hours after LPS challenge, the proventriculi and ceca were collected to analyze CATHs expression. Expression of CATHs was examined at first by reverse transcription-polymerase chain reaction (RT-PCR) using the 0-LPS chicks of non-P-group. The differences in CATHs expression upon probioticsfeeding and LPS were analyzed by real time-PCR. All four CATHs (CATH1, 2, 3 and 4) were expressed in the proventriculus and cecum of chicks. In the proventriculus, the expression of CATHs after LPS challenge did not show significant differences between non-P and P-groups in Experiment 1 and 2. In the cecum, the interactions of the effects of probiotics and LPS on the expression of CATH2 in Experiment 1 and CATH1 and 2 in Experiment 2 were significant, and their expression in 1-LPS chicks was higher in P-group than in non-P-group. However, CATH3 and 4 did not show any significant differences between non-P-and P-groups challenged with LPS. These results suggest that probiotics-feeding may stimulate the immunodefense system mediated by CATH2 and possibly CATH1 against infection by Gram-negative bacteria in the cecum.

The aim of this study was to determine the effects of probiotics-feeding on the gene expression and protein localization of avian β-defensins (AvBDs) in the proventriculus of broiler chicks. Male broiler chicks were arranged in 3 groups: control group, probiotics group I and probiotics group II, which were fed with starter rations containing 0%, 0.2% or 0.4% probiotics, respectively, from day 0 (D0; at one day old) to D14. Proventriculi in all groups were collected at D0, D7 and D14 for analysis of AvBDs expression and AvBD12 protein localization. The expression of AvBDs genes was examined by reverse transcription-PCR and changes in the expression upon probiotics-feeding were examined by real-time PCR. The AvBD12 localization was examined by immunohistochemistry, and density of immunoreaction products was examined by image analysis under a microscope. Out of 14 AvBDs genes, seven AvBDs were detected in the proventriculus of chicks, namely, AvBD1, 2, 4, 6, 7, 10 and 12. The expression of the 7 detected genes did not show any significant differences between control and probiotics groups at D7 and D14. The immunoreactive (ir) -AvBD12 was localized in surface epithelium and cells in the connective tissues of proventricular glands. The ir-AvBD12 density in surface epithelium was significantly higher at D7 than at D0 or D14 in control group. At D7 and D14, the ir-AvBD12 density was significantly lower in probiotics groups than in control group. The ir-AvBD12 cells in proventricular gland increased in number with age; however, there were no significant differences between control and probiotics groups at D7 and D14. These results suggest that, although probioticsfeeding does not affect the gene expression of AvBDs, it may induce AvBD12 secretion from the surface epithelium of the proventriculus in broiler chicks.