Liver Lipid Quantification in NASH and Control Swine at Week 24.

<p>Quantification of lipid species in livers of NASH and control group swine at Week 24 of dietary intervention. Shown is the mean ± standard deviation.</p><p>Liver Lipid Quantification in NASH and Control Swine at Week 24.</p

Liver Lipid Quantification in NASH and Control Swine at Week 24.

<p>Quantification of lipid species in livers of NASH and control group swine at Week 24 of dietary intervention. Shown is the mean ± standard deviation.</p><p>Liver Lipid Quantification in NASH and Control Swine at Week 24.</p

Quantification of hepatic apoptosis at week 24.

<p><b>A:</b> TUNEL assay was performed on paraffin embedded sections. The NASH diet group had significantly higher apoptotic index (number of TUNEL positive cells per 1000 cells) (p = 0.0125). <b>B:</b> Hepatic caspase 3/7 activity shown in fluorescence arbitrary units. Caspase 3/7 activity is significantly higher in NASH diet group than the control diet fed group (p = 0.0397). All data was presented as Mean ± SEM. * indicates statistically significance difference between the NASH diet and the control diet groups.</p

Liver histology by electron microscopy (EM) (A-D 21,400X).

<p><b>A and C:</b> An EM section from a control swine hepatocyte at week 24 shows no accumulation of abnormal material. <b>B and D</b>: An EM section of the liver from a swine fed NASH diet at week 24. Hepatocytes were filled with electron-dense material arranged as whorled lamellar structures, located within membrane-bound vesicles (white arrows) consistent with autophagolysosomes.</p

Liver total, free, and esterified cholesterol measurement and their correlation with liver caspase 3/7 activity at week 24.

<p>Total cholesterol <b>(A)</b>, free cholesterol <b>(B)</b> and esterified cholesterol <b>(C)</b> were higher in the NASH diet fed group than control group. Positive correlations were observed between caspase 3/7 activity and total cholesterol (R² = 0.3770, p = 0.0338) <b>(D)</b> and free cholesterol (R² = 0.4961, p = 0.0105) <b>(E)</b> levels, but not esterified cholesterol (R² = 0.0098, p = 0.7593) <b>(F)</b>. * indicates statistically significance difference between the NASH diet and the control diet groups.</p

Correlation of hepatic free Fatty acid and triglyceride levels with apoptosis at week 24.

<p><b>A:</b> Hepatic free fatty acid levels were more than 5-fold higher in the NASH diet group than control group although it did not reach statistical significance (p = 0.0667). <b>B:</b> There was no difference in hepatic triglyceride concentration between two groups (p = 0.2134). <b>C</b>: A positive correlation was observed between caspase 3/7 activity and fatty acid levels (R² = 0.3096, p = 0.0603). <b>D:</b> There was no correlation between hepatic triglyceride concentration and hepatic caspase 3/7 activity (R² = 0.0684, p = 0.4115).</p

Liver histology of representative control diet (control swine # 957) and NASH diet swine (NASH diet # 954 and 943).

<p>For each animal, top row is H&E and bottom row is trichrome staining; the columns are weeks 8, 16 and 24, respectively. All sections are at 200x magnification except for all week 24 H&E staining, and week 8 and 16 for NASH 943 which are 400x magnification. Control 957 swine showed no evidence of liver injury or fibrosis throughout the experiment. Livers from swine fed NASH diet showed progressive hepatocyte ballooning degeneration (as evidenced by wispy clear cytoplasm), Kupffer cell accumulation, and fibrosis (indicated by an arrow).</p

Histologic features of NASH in serial liver biopsies of both groups of swine.

<p>¶Several percutaneous liver biopsy specimens from baseline were gelatinous and bloody precluding adequate examination</p><p>*One swine in the control group at week 8 and another swine in the control group at week 24 showed <5% hepatocyte ballooning. The NASH diet group demonstrated progressive histological changes of NASH. In the NASH group, feathery and enlarged hepatocytes, consistent with hepatocyte ballooning, started to appear at week 8 and became more prominent at weeks 16 and 24. By week 24, five out of six swine receiving NASH diet exhibiting extensive hepatocyte ballooning (>90% of the hepatocytes). Kupffer cell vacuolization was first observed at week 8, and it became progressively more prominent at weeks 16 and 24. Pericellular fibrosis was first evident at week 16 and it became more prominent at week 24. At week 16, 3 out of 6 swine in the NASH diet group exhibited mild fibrosis and by week 24, 5 out of 6 swine showed moderate fibrosis. Of note, the NASH diet group demonstrated no evidence of significant macrovesicular steatosis or lobular inflammation throughout the dietary intervention. Liver histology of swine in the control group remained unchanged throughout the intervention period.</p><p>Histologic features of NASH in serial liver biopsies of both groups of swine.</p