Franny Jongbloed

132 CHAPTER 5 DR was used as a negative control group while previous studies showed that this dietary intervention did not protect against IRI 17 . Mice on three days of 30% DR were given 70% of their normal daily intake of the SDS chow. Three days of protein-free diet was used as a positive control since this diet protects against IRI 12 . Mice on protein-, Trp-free, Met- free and Leu-free diets were transferred to clean cages with the specific diet given at 4:00 pm. Mice on the control diet were used as the control group. Body weight of the mice was recorded daily. An overview of the composition of the diets used is shown in Table S2. Hepatic ischemia-reperfusion model Mice were anesthetized by isoflurane/N 2 /O 2 inhalation. To maintain their body temperature mice, were placed on a heating pad. All surgeries were performed between 9:00 am and 1:00 pm. Partial (70%) hepatic IRI was induced by occlusion of the blood flow of the left lateral and median liver lobes with a non-traumatic microvascular clamp for 75 minutes, which causes ischemia of the liver tissue. After clamp removal, the restoration of blood flow in the liver leads to reperfusion. No mortality was observed associated with this amount of damage to the liver. After surgery, all mice received 0.5 mL of phosphate-buffered saline subcutaneously and were placed under a heating lamp until they recovered from anesthesia. Directly after, all mice had free access to SDS chow and water. Hepatocellular injury Preoperatively fed and fasted mice were euthanized before surgery (baseline), six hours and 24 hours after reperfusion (n = 5-6 per time point). Serum levels of hepatic damage markers alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) at the Central Clinical Chemical Laboratory of the Erasmus University Medical Center. Hemorrhagic necrosis was scored before, and at six and 24 hours after reperfusion (n = 5-6 per time point) in 3µm thick Hematoxylin and Eosin stained liver sections at a magnification of 100x by two observers blinded to the treatment. Hemorrhagic necrosis was characterized by the loss of the cellular architecture and the presence of erythrocytes in necrotic areas. The percentage of hemorrhagic necrosis per microscopic field was determined using the following scoring system: 0% (absent), 0% to 20% (<20% necrosis per microscopic field), 20% to 40% (20% to <40% necrosis per microscopic field) etcetera until 100% necrosis.