Harvest tissue source does not alter the protective power of stromal cell therapy after intestinal ischemia and reperfusion injury

Abstract

Background Transplantation of mesenchymal stromal cells (MSCs) may be a novel treatment for intestinal ischemia. The optimal stromal cell source that could yield maximal protection following injury, however, has not been identified. We hypothesized that: 1) MSCs would increase survival and mesenteric perfusion, preserve intestinal histological architecture, and limit inflammation following intestinal ischemia and reperfusion injury (I/R), and 2) MSCs harvested from different sources of tissue would have equivalent protective properties to the intestine following I/R. Methods Adult male mice were anesthetized and a midline laparotomy performed. The intestines were eviscerated, the small bowel mesenteric root identified, and baseline intestinal perfusion was determined using Laser Doppler Imaging (LDI). Intestinal ischemia was established by temporarily occluding the superior mesenteric artery for 60 minutes with a non-crushing clamp. Following ischemia, the clamp was removed and the intestines were allowed to recover. Prior to abdominal closure, 2 × 106 human umbilical (USCs), bone-marrow (BMSCs) derived MSCs, or keratinocytes in 250μl of phosphate-buffered saline (PBS) vehicle were injected into the peritoneum. Animals were allowed to recover for 12 or 24 hours (perfusion, histology, inflammatory studies), or 7 days (survival studies). Survival data was analyzed using log rank test. Perfusion was expressed as percentage of baseline and 12 and 24 hour data was analyzed using one way ANOVA and student’s t-test. Non parametric data was compared using Mann-Whitney-U test. A p-value of less than 0.05 was significant. Results All MSCs increased seven day survival following I/R and were superior to vehicle or keratinocytes (P<0.05). All MSCs increased mesenteric perfusion above vehicle at 12 and 24 hours following injury (P<0.05). All MSCs provided superior perfusion compared to keratinocytes at 24 hours post-injury (P<0.05). Administration of each MSC line improved intestinal histology after I/R (P<0.05). Multiple pro-inflammatory chemokines were down-regulated following application of MSCs suggesting a decreased inflammatory response following MSC therapy. Conclusion Transplantation of MSCs following intestinal I/R, irrespective of source tissue, significantly increases survival and mesenteric perfusion while limiting intestinal damage and inflammation. Further studies are needed to identify the mechanism that these cells utilize to promote improved outcomes following injury.

Hydrogen Sulfide Donor GYY4137 Acts Through Endothelial Nitric Oxide to Protect Intestine in Murine Models of Necrotizing Enterocolitis and Intestinal Ischemia

“…The I/R model was carried out on separate adult mice as previously described [10,23,24]. Adult mice (n=6 per group) were anesthetized with 3% isoflurane and maintained under anesthesia with 1.5% isoflurane in oxygen.…”

“…Terminal ileum was acquired and processed for proteins as previously described [10,11,24,25]. Tissue was snap frozen in liquid nitrogen and stored at −80°C.…”

Inhibiting hydrogen sulfide production in umbilical stem cells reduces their protective effects during experimental necrotizing enterocolitis

“…Total volume of each injection was 10µL. The dose is weight-based, modified from our previous work in ischemia-reperfusion [4]. Pups were excluded if they died within the first 24 hours.…”

“…Umbilical mesenchymal stem cells (USC) are isolated from the umbilical cord and are considered multipotent [3]. They have been used with success in animal models of ischemia and reperfusion [4] as well as NEC [5,6]. Stem cells are helpful in repairing tissue through multiple mechanisms including migration and engraftment, heterotopic cell fusion, and paracrine effects including exosome release [3].…”

Direct peritoneal resuscitation improves mesenteric perfusion by nitric oxide dependent pathways

“…Based on our previous work, we learned that MEM conveyed superior survival benefits compared to either plain (serum free, glutamine free, antibiotic free) media or PBS when used as a dialysate during DPR [4]. Previous work also showed a correlation between increased survival and improved mesenteric perfusion [15,16]. Therefore, it stood to reason that the survival benefits to MEM would also be related to improved perfusion.…”