Materials+and+Methods

This multi-part research study looked at a variety of results due to use of the anti-diabetic drug metformin. The mice used for this study were p48 Cre/+ .LSL-Kras G12D/+ mice. This notation means that there is a mutation on the p48 cancer suppressor gene [?, explain p48] that is now "Cre-activated". Cre recombinase is an enzyme that uses site specific recombination to cut and subsequently repair the DNA so that the mutation on p48 is turned on. An adenovirus delivers DNA into the host that codes for the cre recombinase enzyme. This virus is given to transgenic mice in order to induce the activation of the specific mutation needed (Green 2013). The second part of the mouse type denotes with the "lock-stop-lock" (LSL) notation how the gene is engineered. The Kras refers to a specific mutation in Ras called K, with an amino acid change from glycine to aspartic acid at position 12 on the chromosome (Green 2013). PCR was performed for both Kras and Cre genes to determine recombination yields which resulted in 550- and 350bp respectively (Mohammad, et al. 2013). The mice were all fed the control diet for 1 week starting at age 5 weeks old after being separated into separate testing groups so that average body weight for each group was the same. Then, at 6 weeks old, the mice were given their respective treatments (positive control diet, 1000ppm, or 2000ppm metformin dosages). At 44 weeks of age (after 38 weeks of experimental diets), all mice were killed using CO 2 asphyxiation then all pancreata were flash frozen to be used in testing (Mohammad et al. 2013).

IHC (immunohistochemistry) and immunofluorescence analyses were performed on samples of pancreatic tissue to determine the effects on mTOR and pAMPK in tumor cells (Mohammad et al. 2013). Small sections were incubated for 1 hour with primary antibodies staining for mTOR and pAMPK. Secodary antibodies of anti-mouse or anti-rabbit (depending on the primary) were used to incubate for 1 hour more. DAB was used to visualize results and the pancreatic cells were further stained with hematoxylin for IHC and with DAPI (for DNA material) for IHF. Cells were visualized with microscope then photos taken (Mohammad et al. 2013).

Presence of PanIN lesions, types 1, 2, and 3 were determined in order to establish a relationship between metformin and PanIN multiplicity. A negative control of wild-type mice and a positive control of transgenic mice only given a control diet (no metformin added) were use to test against the two experimental conditions: 1000ppm and 2000ppm dosages of metformin (Mohammad et al. 2013). All samples of pancreatic tissues were fixed with 10% neutral buffered formalin for 24-48 hours, processed, and embedded in paraffin in order to be stained with hematoxylin and eosin. To quantify the progression of PanIN lesions, grades (1, 2, or 3) and numbers of ductal lesions were determined for each sample. These samples were also analyzed for spread of PDAC, indicative of metastatic pancreatic cancer (a later, more severe case of the cancer). Normal pancreatic tissue was also looked at to determine a baseline for that individual, should any other abnormalities occur. Data was then further tested statistically with an unpaired t-test (Mohammad et al. 2013).