CD-1 male mice, 30 g body weight, were maintained in light-dark cycles of 12 hours. They received a laboratory chow diet (CD) containing 18% protein, 5% fat and 5% fiber. The food was powdered, and the statin was added in the amounts mentioned ahead and administered orally by different periods of time as indicated forward. The hypercholesterolemic diet (HD) contained 2% cholesterol and 0.6% sodium deoxycholate. Rosuvastatin 20 mg tablets were obtained from Medimart, Slovenia. All other compounds were purchased from Merck-Sigma Mexico.
Each part of the experiment included animals with CD and HD alone (n = 6). The experimental procedures were managed according to the Official Mexican Standard NOM-062-ZOO-1999 and the guidelines established by the Research Committee for the Care and the Use of Laboratory Animals of the Universidad Nacional Autónoma de México.
A) The animals received a continuous treatment with rosuvastatin (Ro) in order to evaluate the survival rate:
CD + Ro (rosuvastatin 0, 20, 50, 100, 200, 400 mg/Kg/day)
HD + Ro (rosuvastatin 0, 20, 50, 100, 200, 400 mg/Kg/day
B) The animals received a continuous treatment with moderate high statin doses to evaluate hepatocyte mitochondrial respiration. The microscopic observations were performed in representative samples of each group after different treatment lengths.
CD + Ro (rosuvastatin 0, 20 mg/Kg/day)
HD + Ro (rosuvastatin 0, 20 mg/Kg/day)
C) The animals received a continuous treatment with median statin doses to evaluate hepatocytes mitochondrial respiration and to perform microscopic observations in representative samples of each group after different treatment lengths:
HD + Ro (rosuvastatin 0, 5 mg/Kg/day)
HD + Ro (rosuvastatin 0, 2.5 mg/Kg/day)
HD + Ro (rosuvastatin 0, 1 mg/Kg/day)
The animals were sacrificed by decapitation. Blood and other tissues were immediately obtained for biochemical analysis or microscopic studies.
Mitochondria isolation and incubation
Liver was homogenized in 250 mM sucrose, 0.5 mM HEPES, 0.5 mM EGTA (SHE). pH 7.2, using a Thomas pestle tissue grinder (piston-type Teflon pestle) and mitochondria were isolated employing a refrigerated centrifuge MPW-353R Med Instruments, Varsovia, following the method described by Frezza et al., . The mitochondria obtained by centrifugation were incubated for 10 min with 0.5% albumin to eliminate fatty acids and resuspended in SHE solution. Protein content in mitochondria was evaluated by the Bradford method . The incubation media contained: KCl 240 mM, HEPES 60 mM, H3PO4 4 mM, EGTA 4 mM, Succinate 10 mM, MgCl2 4 mM, mitochondrial protein 4 mg, pH 7.2, final volume 3.2 ml. An YSI oxygen meter 5300 model was employed to measure the oxygen consumption. The mitochondrial respiration was stimulated by the addition of 10 µL of 200 mM ADP and the respiratory control was evaluated.
Serum determinations included total cholesterol, triacylglycerols, HDL-C (high density lipoprotein cholesterol), glucose, urea, creatinine, AST (aspartate aminotransferase) and ALT (alanine aminotransferase), employing a semi-automatic equipment of clinical chemistry from Random Access Diagnostics.
The liver was studied both by light and electronic microscopy. The tissue slices dyed with hematoxylin-eosin were observed in a Nikol Eclipse 180 microscope. For electronic microscopy the mitochondrial pellet and the liver tissue were fixed with 3% glutaraldehyde in cacodylate buffer. The samples were processed in the microscopy unit of the Neurobiology Institute, Universidad Nacional Autónoma de México, employing a Jeole transmission electronic microscope model JEM-1010.
The mice survival was expressed in percent. One-way analysis of variance (ANOVA), followed by Student–Newman–Keuls test and differences were considered to statistical significance when p < 0.05. For morphologic analysis we used representative samples from each of the animal groups.