8 equivalent to 20 or 19.2 mg/kg body weight/day ad libitum until 90 days of age. Mice were genotyped for the ApcMin allele as reported. All protocols Calcium Channel cancer were approved by the UNC Institutional Animal Care and Use Committee. Intestinal tumor analysis At three months of age, B6 ApcMin/ mice were euthanized and gastrointestinal tracts from pylorus to rectum were removed. The small intestine was cut into thirds, and the caecum and colon were separated. Segments were gently flushed with PBS to remove fecal material, cut longitudinally, splayed flat on Whatmann 3MM paper and fixed overnight at 4 in 4% paraformaldeyhyde. Polyps were counted and their diameters measured using a dissection microscope with an in scope micrometer, allowing detection of polyps greater than 0.3 mm in diameter.
Echocardiography Tosedostat LPA receptor inhibitor Transthoracic echocardiography was performed at baseline and prior to sacrifice using a 30 mHz probe on a Vevo 660 Ultrasonograph. B6 wild type mice were lightly anaesthetized with 1 1.5% isofluorane and a topical depilatory agent applied before placing in the left lateral decubitus position under a heat lamp to maintain body temperature at 37. Heart rate was maintained between 450 to 500 beats per minute. Two dimensional short and long axis views of the left ventricle were obtained. M mode tracings were recorded and used to determine left ventricle end diastolic diameter, LV end systolic diameter, LV posterior wall thickness diastole and LV posterior wall thickness systole over three cardiac cycles. LV fractional shortening was calculated using the formula % FS /.
All measurements were performed by two independent observers blinded to the treatment group. Barrick et al. Page 3 Toxicol Appl Pharmacol. Author manuscript, available in PMC 2009 May 18. NIH PA Author Manuscript NIH PA Author Manuscript NIH PA Author Manuscript Histology At necropsy, hearts, lungs, liver and kidneys were dissected from treated and control B6 wildtype mice, rinsed in PBS and weighed. Hearts were cut in cross section just below the level of the papillary muscle. For assessment of cardiomyocyte size, cardiac cell apoptosis and fibrosis, the top half of the heart was formalin fixed and embedded in paraffin. Sections were prepared at 200 m intervals. The sections were stained with hematoxylin and eosin for examination of gross appearance, aortic valve size and cardiomyocyte size, while Masson,s Trichrome was used to facilitate visualization of fibrosis.
Sections were included for measurement of aortic valves only when the aortic outflow tract was clearly visible and parallel to the plane of sectioning and the entire cross section of two valve leaflets was visible and could be clearly traced to the attachment point. Cardiomyocyte hypertrophy was assessed by measuring cross sectional area of 100 cardiomyocytes per periodic acid Schiff hematoxylin stained section in ten randomly selected fields having nearly circular capillary profiles and centered nuclei in the left ventricular free wall. Histological images were analyzed using Nova Prime 6.75.10 software. Apoptotic cells were identified in serial cardiac cross sections with the ApopTag Fluorescein In Situ apoptosis detection kit according to the manufacturer,s protocol. Images were acquired on a Nikon E800 microscope with a Hammamatsu ORCA ER charge coupled device camera with Metamorph software and processed with Photoshop.