T. D. Crafts1, E. Blocher-Smith1, T. A. Markel1 1Indiana University School Of Medicine,Pediatric Surgery,Indianapolis, IN, USA
Introduction: Cellular therapy is a novel surgical treatment option for intestinal ischemia. Surgeons have direct access to injured bowel at the time of laparotomy and could use the intraperitoneal application of stem cells to salvage necrotic or marginal appearing intestine. Bone Marrow Derived Mesenchymal Stem Cells (BMSCs) have previously been shown to abate the damage caused by intestinal ischemia/reperfusion injury (I/R) We hypothesized that: 1) application of human BMSCs to intestine following I/R would improve survival, and 2) improved outcomes would be associated with increased tissue levels of angiogenic and intestinal growth factors, namely IL-6, VEGF, and CXCL-10.
Methods: Adult BMSCs were cultured on polystyrene flasks in alpha-MEM containing fetal bovine serum (37C, 5%CO2 in air). Cells were subcultured or used for experimentation at 90% confluence between passages 3-10. Eight week old male C57Bl6J mice (20-25g) were then anesthetized and underwent a midline laparotomy. I/R and MSC groups were exposed to superior mesenteric artery ligation for 60 minutes with a non-traumatic clamp. Immediately following removal of the clamp, 2 x 106 human BMSCs in PBS were placed into the abdominal cavity. Animals were then closed in two layers and allowed to reperfuse for 6 hours (molecular analysis) or 7 days (survival analysis). Following 6 hour reperfusion, animals were euthanized. Intestines were harvested and homogenized in RIPA buffer with phosphatase and protease inhibitors. Extracts were quantified for total protein content (Bradford Assay) and analyzed by multiplex beaded assay for IL-6, VEGF, and CXCL10. P<0.05 was significant.
Results: I/R caused marked intestinal ischemia and significant mortality. Seven day survival was 30% for I/R, while application of MSCs following ischemia increased the seven day survival to 80% in a dose dependent fashion (p<0.05, Figure 1A). MSC application increased intestinal tissue levels of IL-6 (IR: 39.6+/-5.2 ng/g protein, MSC: 84.3+/-19.2 ng/g protein, p=0.03) and CXCL10 (IR: 40.4+/-4.4 ng/g protein, MSC: 52.5+/-3.5 ng/g protein, p=0.03) but not VEGF (IR: 20.0+/-1.5 ng/g protein, MSC: 22.4+/-3.0 ng/g protein, p=0.25)(Figure 1B-D).
Conclusion: Direct application of human BMSCs to the peritoneal cavity following intestinal I/R improved survival by fifty percent. Improved outcomes were associated with higher intestinal tissue levels of IL-6 and CXCL10, but not VEGF. Further studies are needed to elucidate stem cell mechanisms in order to harness maximum therapeutic potential.
