Animals and experimental protocol

Animals and experimental protocol selleck products FVB mice of both gender were obtained from Harlan Inhibitors,Modulators,Libraries Win kelmann and used at 6 8 weeks of age. The animals were fed standard mouse chow and were allowed to take food and water ad libidum. All experi ments conformed to the NIH guidelines to the care and use of experimental animals, and were approved by the local authorities. The kinetic of proliferation within the walls of intrapul monary vessels in response to reduced oxygen supply was examined in mice kept for 2, 3, 4, 10, 16, or 21 days in a hypobaric chamber. An air intake valve was adjusted to maintain intrachamber pressure at 380 mmHg while allowing adequate airflow through the chamber to prevent accumulation of CO2 and NH3. Control mice were kept at normobaric pressure at room air.

To examine the effect of Inhibitors,Modulators,Libraries rapamycin on hypoxia induced vascular remodeling and right ventricular hypertrophy, age matched mice were divided into 6 experimental groups 1. untreated normoxic mice, 2. vehicle treated normoxic mice, 3. rapamycin treated normoxic mice, 4. untreated Inhibitors,Modulators,Libraries hypobaric mice, 5. vehicle treated hypobaric mice, and 6. rapamycin treated hypobaric mice. In some experiments solely four groups were formed. For application of rapamycin or vehicle, the chamber was opened daily and the mice were weighed. An 1. 75 mg/ml stock solution of rapamycin was freshly prepared every second day by homogenization of the drug in 0. 2% car boxymethylcellulose as vehicle. Rapamycin was injected i. p. at 3 mg/kg d in a final Inhibitors,Modulators,Libraries volume of 100l. Control mice received either the same volume of the vehicle or remained untreated.

Tissue preparation Mice were sacrificed by cervical dislocation and exsan guinated by cutting the vena cava inferior. The chest cavity was opened, and the lungs were filled via the trachea with Zamboni fixative. Heart and lungs were removed en block and transferred into Zamboni Inhibitors,Modulators,Libraries fix ative. After fixation for 6 h, the tissue was washed over night with 0. 1 mol/L phosphate buffer and incubated for 3 days with increasing concentrations of sucrose solution. Finally, the specimens were embedded in optimal cutting temperature compound and frozen in liquid nitrogen. Immunohistochemistry Immunohistochemical double labeling of lung tissue for Ki67 and smooth muscle actin was employed for a quantitative analysis of the proliferative activity of the pulmonary vas culature.

For that purpose, 10m thick frozen sections were prepared and Ki67 antigen any other enquiries was unmasked by micro wave treatment. After blocking of unspe cific protein binding sites, the frozen sections were incu bated overnight simultaneously with FITC conjugated anti smooth muscle actin antibody and anti Ki67 anti body followed by Cy3 conjugated don key anti rabbit antibody. After three washes with PBS the sections were incubated with 1g/ml DAPI in PBS for 15 min followed by three washes with PBS.

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