All experiments were approved by the Cincinnati Children’s Hospital Institutional Animal Care and Use Committee (IACUC). 8–10 week-old female FVB/NJ mice (Jackson Laboratory, Bar Harbor, ME; Stock Number, 001800) were anesthetized using isoflurane and then shaved with an electric shaver so as to avoid injury (Oster, McMinnville, TN). Shaved mice were cleaned with both betadine® surgical scrub (Purdue Products L.P., Stamford, CT) and isopropyl alcohol (Vedco, Inc., Saint Joseph, MO) prior to creating 8mm diameter, full thickness, circular wounds on bilateral flanks of each mouse. The skin wounds were then covered with a sterile transparent dressing (Tegaderm; 3M Healthcare, St. Paul, MN) before the mice were housed individually for recovery. Non-wounded but anesthetized, shaved, and bandaged mice were also utilized for comparison.
At the conclusion of the time course, mice were anesthetized using isofluorane and PB was collected from the retro-orbital space into EDTA coated tubes (BD, San Jose, CA). Following sacrifice by cervical dislocation, bilateral femurs were harvested into ice cold PBS. One femur was flushed with 3mL ice-cold PBS using a 21-gauge needle for flow cytometric analysis and RNA isolation, while the other was flushed with 40µL of ice-cold PBS containing 1% Igepal, 8µL protease inhibitor cocktail, 20mM NaF, and 1mM Na3VO4 (all from Sigma-Aldrich Co., St Louis, MO) for protein isolation. Femurs from one mouse per group were harvested into 10% neutral buffered formalin at room temperature (RT) for 24 hours for immunohistochemistry. Whole blood was centrifuged to collect plasma for SDF-1α quantitation. All samples were then stored at −80°C until use.
Immunostaining and flow cytometry analyses were performed according to standard procedures, and all cells were analyzed on a FACSCanto II flow cytometer (BD Biosciences). To label EPCs in PB and BM, cells were stained using anti-CXCR4 (biotinylated, clone 2B11, BD Bioscienes), anti-Sca-1 (PE-Cy7 conjugated, clone D7), anti-c-Kit (APC-AF750 conjugated, clone 2B8), anti-CD133 (PE conjugated, clone 13A4), and anti-Flk-1 (APC conjugated, clone Avas12A1) monoclonal antibodies followed by streptavidin FITC (all from eBioscience, San Diego, CA). For live/dead discrimination, cells were resuspended in medium containing 7-AAD (eBioscience) prior to analysis. Each data point included at least 100,000 events. Flow data were then analyzed using FlowJo software (Tree Star Inc., Ashland, OR)
Real time reverse transcription-PCR
Total RNA was isolated from bone marrow using the RNeasy mini kit (Qiagen Inc.,Valencia, CA) per manufacturer’s recommendations. Following cDNA conversion, SDF-1α expression was analyzed using an Applied Biosystems 7300 Real-Time PCR System (Applied Biosystems, Foster City, CA) with forward primer 5’-GGT TCT TCG AGA GCC ACA TCG-3’ and reverse primer 5’-ACG GAT GTC AGC CTT CCT CG-3’. SDF-1α expression was normalized to beta-2-microglobulin using the forward primer 5’-GGCCTGTATGCTATCCAGAA-3’ and reverse primer 5’-GAAAGACCAGTCCTTGCTGA-3’. Real time PCR data were analyzed using the 2−ΔΔCT
Enzyme-linked immunosorbent assay (ELISA)
SDF-1α protein levels were determined within BM and plasma samples using the mouse SDF-1 alpha Quantikine ELISA Kit (R&D Systems, Minneapolis, MN) per manufacturer’s instructions. BM samples were then normalized to total protein, calculated using the Bio-Rad Protein Assay (Bio-Rad laboratories, Hercules, CA).
Femurs were removed from 10% formalin after 24 hours, washed briefly in deionized (DI) water and decalcified in immunocal™ solution (Decal Chemical Corporation, Tallman, NY) for 3 days at RT. Femurs were rinsed in DI water, dehydrated through a series of alcohols and cleared with 3 changes of xylenes. Femurs were soaked in three changes of infiltrating paraffin wax, allowed to cool and embedded in paraffin blocks for sectioning. Five micron sections were placed onto SuperFrost Plus slides (Fisher, Pittsburgh, PA) and allowed to dry overnight. Sections were deparaffinized, and rehydrated in graded alcohols. Sections were blocked with 3% hydrogen peroxide in water for 10 minutes followed by a 10 minute Avidin and Biotin block (Dako Inc, Carpinteria, CA). A Protein Block solution (Dako Inc, Carpinteria, CA) was then placed on the sections for 15 minutes at RT. Sections were incubated with rabbit anti-mouse SDF-1α (1:500) (Abcam Inc., Cambridge, MA) for 30 minutes at RT, rinsed in 1X Wash Buffer (Dako Inc, Carpinteria, CA) and incubated with biotinylated goat anti-rabbit (1:200) (Vector Labs, Burlingame, CA) for 30 minutes at RT. After rinsing, tissues were incubated with Elite ABC reagent (Vector Labs, Burlingame, CA) for 30 min at RT. Slides were rinsed and incubated in DAB reagent (Dako Inc., Carpinteria, CA) for 5 min at RT. Sections were counterstained using hematoxylin, dehydrated, cleared, mounted using permount (Fisher, Pittsburgh, PA) and coverslipped.
The flow data were analyzed using the nonparametric Kruskal-Wallis test followed by Wilcoxon rank sum tests for pairwise comparisons. The remaining comparisons were performed using one-way ANOVA followed by Tukey post hoc means comparison test. All of the statistical analyses were carried out using SPSS for Windows (SPSS Inc., Chicago, IL). The data are expressed as the mean ± S.E.M. Differences at p < 0.05 were considered significant.