More detailed information on this methodology has been previously published

onditions at a controlled temperature of 232C, relative humidity 5510%, and with 12 h light/12 h dark cycles. They were given food and water ad libitum. Mice were checked for their health during the entire experimental period at least once a day after tumor injection. All surgery was performed under medetomidine /midazolam /butorphanol anesthesia. Mice were normally euthanized by CO2 inhalation at the end of a study. For the peritoneal dissemination model, Panc02-Luc-ZsGreen cells were injected intraperitoneally as a cell suspension into 7-week-old male C57BL/6 mice, and the mice were randomized and grouped into the control and the SSHE groups. The treatment regimens were started the day after tumor inoculation. Mice were euthanised in a CO2 chamber when they PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19666601 were moribund, measured by a lack of sustained purposeful response to gentle stimuli. All efforts including subcutaneous administration of meloxicam were made to minimize suffering. The experiment was performed twice, and the combined data were subjected to Oleandrin site analyses. For the orthotopic model of pancreatic cancer, Panc-1-Luc-ZsGreen cells were implanted with 50% Matrigel into the pancreas of 6-week-old female nude mice . One week after the injection, they were randomized and grouped into the control and the SSHE groups. The treatment regimens were started one week after tumor injection. For mouse colon carcinoma experiments, LuM1 cells were subcutaneously implanted in male Balb/c mice. The volumes of LuM1 tumors were evaluated by measuring two perpendicular diameters with calipers. Tumor volume was calculated using the following equation: V = /2, where a is the small diameter and b the large diameter. In the SSHE group, mice were intraperitoneally 5 / 22 Antitumor Activity of Ginger Extract against Pancreatic Cancer administered 80 mg/kg SSHE once daily. In the control group, mice were administered solvent alone in DPBS. Bioluminescent imaging In vivo bioluminescent imaging was performed using the IVIS imaging system. All mice were injected intraperitoneally with 150 mg/kg d-luciferin and anesthetized with 2.5% isoflurane. PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19667299 Ten minutes later, photons from animals’ whole bodies were imaged using the IVIS imaging system according to the manufacturer’s instructions. Data were analyzed by living image 2.50 software. Blood hematology and biochemistry test Mice were anesthetized, and blood was collected from the heart. Peripheral blood profiles were analyzed by the Sysmex KX-21NV automated hematology analyzer. The levels of white blood cells, red blood cells, platelets, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration were examined. Glucose levels, total cholesterol levels, alanine aminotransferase and aspartate aminotransferase levels, and blood urea nitrogen levels were analyzed with an automated analyzer for clinical chemistry, SPOTCHEM EZ SP-4430, using SPOTCHEM II test strips. Reversed-phase high-performance liquid chromatography Liquid chromatographic separations were achieved using a reversed-phase C-18, 3 m, 2.4 250 mm column at a flow rate of 1 ml/min. The mobile phase was 70% methanol. The elution profile was monitored by UV spectrophotometry at 228 nm. Statistical analysis All data are presented as the mean SD. Statistical significance between data sets was tested by unpaired Student’s t test. Survival of mice was analyzed using the log-rank test. P < 0.05 was considered to be statistically significant. R