Simvastatin was kindly donated by Merck Co, Inc, USA. MSCs were labeled with 1,1 dioctadecyl 3,3,33 testramethylindo carbocyanine perchlorate before transplantation as described unfortunately previously. Briefly, 2 ug/ml DiI was added to cells suspension and incubated at 37 C for 5 minutes, then at 4 C for 15 min utes with occasional mixing. MSCs labeled with DiI were washed 3 times with PBS and then collected. Laser Doppler blood perfusion analysis The ratio of ischemic/normal hindlimb blood flow was measured using laser Doppler blood perfusion imager as described previously. Low to no flow was displayed as dark blue, whereas high blood flow was displayed as red to white. Previous study has demonstrated that hindlimb blood flow was progressively augmented over the course of 14 days, ultimately reaching a plateau between 21 and 28 days in mouse hindlimb ischemia model.
There fore, at three predetermined time points, we performed 2 consecutive laser scanning over the same region of interest. The average Inhibitors,Modulators,Libraries flow of the ischemic and nonischemic legs was calculated on the basis of histograms of the colored pixels. To mini mize variations due to ambient light, blood flow was expressed as the ischemic /normal limb flow ratio. Histological assessment for capillary density Ischemic limb muscles were harvested at Inhibitors,Modulators,Libraries day 21 after treatment and embedded in optimal cutting temperature compound. Frozen tissue sections of 5 um thick were stained for alkaline phosphatase to examine the capillary density.
To ensure that the capillary densities were not overestimated as a consequence of myocyte atrophy or underestimated because of interstitial edema, the capillary/muscle fiber Inhibitors,Modulators,Libraries ratio was determined. Terminal deoxynucleotidy1 transferase mediated dUTP nick end labeling assay The terminal deoxynucleotidy1 transferase mediated dUTP nick end Inhibitors,Modulators,Libraries labeling assay was performed to determine apoptotic activity in hindlimb ischemic tis sues using an In Situ Cell Death Detection Kit according to the manufacturers instructions. Cells in which the nucleus was stained brown were defined as TUNEL positive and the percentage of apop totic cells per total number of cells was determined by two independent blinded investigators. Western blot analysis for the expression of VEGF protein in vivo Lysates from hind limb muscle tissue homogenates har vested at day 21 post surgery were used for Western blot analysis as described previously.
Protein was analyzed using 10% sodium dodecyl sulphate polyacryla mide gel electrophoresis Inhibitors,Modulators,Libraries and transferred to nitrocellulose membranes. Membranes were then incubated with primary antibodies including VEGF and b actin at 4 C overnight respectively. selleck chem The membranes were then incubated with peroxidase labeled secondary antibody at 37 C for 2 hours. Signals were detected by enhanced chemiluminescence. Densitometric analysis for the blots was performed with NIH image software.